VETERINARY 
BACTERIOLOGY 

A  TREATISE  OX  THE 

BACTERIA,  YEASTS,  MOLDS,  AND  PROTOZOA 
PATHOGENIC    FOR     DOMESTIC    ANIMALS 


BY 

ROBERT    EARLE    BUCHANAN,    Ph.D. 

PROFESSOR      OF      BACTERIOLOGY     IN     THE     IOWA     STATE     COLLEGE 
OF    AGRICULTURE    AND    MECHANIC  ARTS,  DIVISION  OF  VETERINARY 

MEDICINE;     BACTERIOLOGIST    OF     THE     IOWA     AGRICULTURAL 
EXPERIMENT  STATION 


WITH  21 4  ILLUSTRATIONS 


PHILADELPHIA  AND  LONDON 

W.     B.     SAUNDERS     COMPANY 
1911 


Copyright,  ign,  by  W.  B.  Saunders  Company 


PRINTED    IN    AMERICA 

PRESS    OF 

B.     8AUNDER8     COMPANY 
PHILADELPHIA 


PREFACE 


THE  present  volume  is  a  revision  of  the  lectures  on  veterinary 
bacteriology  given  during  the  past  six  years  to  classes  in  the  Division 
of  Veterinary  Medicine  in  the  Iowa  State  College.  It  constitutes 
a  serious  attempt  to  put  in  usable  form  that  fund  of  knowledge 
concerning  bacteriology  which  the  student  of  veterinary  medicine 
should  master.  It  is  in  no  sense  a  text  on  pathology,  and  discus- 
sion of  purely  pathological  subjects  has  been  minimized  as  much  as 
possible.  The  intention  has  been  to  confine  attention  as  far  as 
practicable  to  those  topics  that  unquestionably  lie  in  the  province 
of  bacteriology.  This  has  been  defined  to  include  a  discussion 
of  immunity  and  of  the  pathogenic  bacteria,  yeasts,  molds,  and 
protozoa. 

The  book  is  not  intended  to  serve  as  a  manual  of  laboratory 
practice,  hence  detailed  discussion  of  methods  and  technic  has 
been  omitted.  Methods  of  significance  in  diagnosis  or  treatment 
are  given  in  greater  detail  in  the  discussion  of  specific  organisms. 

Several  organisms  causing  diseases  of  man  not  transmissible 
to  lower  animals  have  been  included.  In  all  cases  they  are  closely 
related  to  organisms  having  significance  to  the  veterinarian,  they 
cause  diseases  which  are  commonly  confused  with  somewhat  similar 
diseases  of  lower  animals,  or  they  are  valuable  as  illustrations 
of  methods  of  immunization,  treatment,  or  diagnosis.  Such 
organisms  are  relatively  few  in  number. 

A  group  system  of  discussion  of  the  pathogenic  bacteria  has 
been  adopted.  The  classification  used  has  proved  very  helpful  in 
my  own  classwork.  The  groupings  used  are  not  entirely  satis- 
factory, in  part  due  to  the  fact  that  some  of  the  species  have  not 
been  adequately  described  and  differentiated  in  the  literature. 
An  effort  has  been  made  to  point  out  the  deficiencies  in  our  present 
knowledge,  both  to  give  a  better  balanced  presentation  of  the  sub- 
ject and  to  stimulate  interest  in  the  solution  of  the  problems. 

222584,  9 


10  PREFACE 

The  pathogenic  protozoa  constitute  a  group  which  is  particu- 
larly difficult  to  treat  adequately,  largely  due  to  the  rapid  growth 
of  the  subject.  Relatively  few  of  the  forms,  moreover,  are  of 
immediate  interest  to  the  North  American  student. 

I  wish  gratefully  to  acknowledge  the  assistance  of  my  wife  in 
the  preparation  of  illustrations  and  revision  of  manuscript,  that 
of  Mr.  Chas.  Murray  in  proof  reading,  and  that  of  Dean  Chas.  C. 
Stange  and  Dr.  Murphy,  of  the  Division  of  Veterinary  Medicine, 
for  critical  reading  of  the  manuscript. 

R.  E.  BUCHANAN. 

IOWA  STATE  COLLEGE,  AMES,  IOWA. 
July,  19 11-. 


CONTENTS 


SECTION  I 

MORPHOLOGY,   PHYSIOLOGY,   AND  CLASSIFICATION   OF 

BACTERIA 
CHAPTER  I. — INTRODUCTION 17 

The  Microscope  and  Its  Influence,  18. — Nature  and  Classification  of  Microor- 
ganisms, 20. — Spontaneous  Generation,  20. — Relation  of  Microorganisms  to 
Fermentation  and  Decay,  21. — Relationship  of  Microorganisms  to  Disease,  21. — 
Development  of  Laboratory  Methods,  22. — Development  of  Theories  of  Immun- 
ity, 23. — Development  of  Sanitary  Science  and  Preventive  Medicine,  23. 

CHAPTER  II. — MORPHOLOGY  AND  RELATIONSHIPS  OF  MICROORGANISM- 

CONCERNED  IN  DISEASE  PRODUCTION 25 

Position  of  Pathogenic  Microorganisms,  25. — Differentiation  of  Animals  and 
Plants,  25. — Subdivisions  of  the  Thallophytes,  26. — Morphology  of  Bacteria,  27. — 
Shape  of  Bacteria,  27. — Grouping  of  Bacterial  Cells,  28. — Size  of  Bacteria,  30. — 
Histology  and  Structure  of  Bacteria,  31. — Reproduction  in  Bacteria,  35. — Mor- 
phology of  the  Yeasts,  Saccharomycetes,  and  Blastomycetes,  37. — Form,  Size,  and 
Grouping  of  Yeasts,  38. — Histology  and  Structure  of  the  Yeast,  38. — Yeast 
Protoplasm  and  Cell  Inclusions,  38. — Reproduction  in  Yeasts,  39. — Morpholoau 
of  the  Hyphomycetes  or  Molds,  40. — Form  and  Size  of  Hyphomycetes  or  Molds,  40. 
— Histology  and  Structure  of  Molds,  41. — Reproduction  of  Molds,  42. — Morphol- 
ogy of  the  Protozoa,  43. — Form  and  Size  of  Protozoa,  44. — Histology,  44. — Repro- 
duction, 44. 

CHAPTER  III. — PHYSIOLOGY  OF  MICROORGANISMS 45 

Food  Relationships  of  Microorganisms,  45. — Composition  of  the  Cell,  45. — Sources 
and  Kinds  of  Foods,  45. — Moisture  Relationships  of  Microorganisms,  46. — Respir- 
ation of  Microorganisms,  47. — Temperature  Relationships  of  Microorganisms,  48. — 
Optimum  Temperature,  48. — Minimum  Temperature,  48. — Maximum  Growth 
Temperature,  48.— Growth  Temperature  Range,  48. — Thermal  Death  Point,  48. 
— Light  Relationships  of  Microorganisms,  49. — Effect  of  Electricity  on  Bacteria, 
50. — Relationships  of  Microorganisms  to  Chemicals,  50. — Chemotaxy,  50. — Tro- 
pisms,  51. — Influence  of  Reaction  of  Medium  on  Growth,  52. — Antiseptics  a»>l 
Disinfectants,  52. — Disinfectants  and  Antiseptics  in  Common  Use,  53. — Adjust- 
ment of  Organisms  to  Osmotic  Pressure,  55. — Symbiosis,  Antibiosis,  and  Com- 
mensalism,  56. — Pigment  Production  by  Microorganisms,  56. — Light  Production  by 
Microorganisms,  57. — Fermentation  and  Enzyme  Production,  58. 


CHAPTER  IV. — CHANGES  OF  ECONOMIC  SIGNIFICANCE  BROUGHT  ABOUT 

BY  NON-PATHOGENIC  ORGANISMS 61 

Production  of  Alcohol,  62. — Production  of  Acids,  62. — Decay  and  Putrefaction,  64. 
— Reduction  Processes  in  Inorganic  Compounds,  66. — Oxidation  of  Inorganic 
Compounds,  67. — Miscellaneous  Changes.  74. 

CHAPTER  V. — CLASSIFICATION  OF    MICROORGANISMS 75 

Classification  of  Bacteria,  76. — Key  to  the  Groups  and  Genera  of  Bacteria,  77. — 
Streptococcus,  77. — Diplococcus,  78. — Micrococcus,  78. — Staphylococcus,  78. — 
Bacillus,  78. — Spirillum,  79.— Spiroehseta,  80. — Chlamydobacteriacea?,  80. — 
Beggiatoa,  SI. — Classification  of  Yeasts,  82. — Classification  of  the  Molds,  82. 

11 


12  CONTENTS 

SECTION  II 

LABORATORY    METHODS   AND   TECHNIC 

PAGE 

CHAPTER  VI. — STERILIZATION 83 

Sterilization  by  the  Flame,  SS.-rSterilization  by  Hot  Air,  83.— Sterilization  by 
Streaming  Steam,  84. — Sterilization  by  Steam  under  Pressure,  85. — Sterilization 
at  Temperature  Lower  than  Boiling-point,  87. — Sterilization  by  Addition  of 
Chemicals,  87. — Sterilization  by  Filtration,  87. 

CHAPTER  VII. — CULTURE-MEDIA  AND  THEIR  PREPARATION 89 

Use  of  Normal  Salt  Solutions  of  Acid  and  Alkali  and  Methods  of  Expressing 
Reactions,  89. — Nature  of  Nutrients  Required  by  Bacteria,  90. — Liquid  Media, 
91. — Bouillon  or  Beef  Broth  from  Meat.  91. — Bouillon  or  Broth  from  Beef  Extract, 
91. — Sugar-free  Broth,  91. — Sugar  Broth,  92. — Glycerin  Broth,  92. — Serum  Broth, 
92.— Dunham's  Solution,  92.— Beerwort,  92.— Milk,  92.— Synthetic  Media,  92.— 
Liquefiable  Solid  Media,  93. — Nutrient  Gelatin,  93. — Other  Gelatin  Media,  93. — 
Nutrient  Agar,  93.— Other  Agar  Media,  93.— Non-liquefiable  Media,  94.— Potato, 
94.— Other  Vegetable  Media,  94.— Blood-serum,  94.— Egg  Medium,  95. 

CHAPTER  VIII.— BIOCHEMICAL  TESTS 96 

Acid  Production,  96. — Alkali  Production,  96. — Gas  Production,  96. — Reduction 
Processes,  97. — Indol  Production,  98. — Thermal  Death-point,  99. — Efficiency  of 
Disinfectants,  99. 

CHAPTER  IX. — MICROSCOPIC  EXAMINATION  AND  STAINING  METHODS.  .    100 

Measuring  Bacteria,  101. — Examining  of  Living  Bacteria,  101. — Hanging  Drops, 
101. — Staining  Methods,  102. — Mordants,  102. — Formulas  of  Some  of  the  Com- 
monly Used  Stains,  102.— Preparation  of  a  Stained  Mount,  103.— Spore  Stain,  104. 
— Stain  for  Acid-fast  (Acid-proof)  Organisms,  104. — Flagella  Stain,  105. — 
Gram's  Staining  Method,  106.— Blood  and  Protozoan  Stains,  106. 

CHAPTER  X. — METHODS  OF  SECURING  PURE  CULTURES  OF  BACTERIA.  . . .   107 

Dilution  Method,  107. — Isolation  by  Smearing,  107. — Direct  Isolation,  107. — 
Isolation  by  Plating,  108.— Isolation  by  the  Use  of  Heat,  109.— Isolation  by  the 
Use  of  Differential  Antiseptics  or  Disinfectants,  109. — Isolation  by  Animal  Inocu- 
lation, 109. 

CHAPTER  XI. — STUDY  OF  BACTERIAL  CULTURES 110 

Cultural  Characters,  110. — Agar  Stroke,  110. — Potato,  110. — Blood-serum,  111. — 
Gelatin  Stab,  111.— Nutrient  Broth,  111.— Milk,  111.— Litmus  Milk,  112.— 
Gelatin  Plate  Colonies,  112. — Colonies  on  Agar  Plates,  113. — Physiological 
Characters,  115. 


SECTION  III 

BACTERIA  AND  THE  RESISTANCE  OF  THE  ANIMAL  BODY  TO 

DISEASE 

CHAPTER  XII. — BACTERIA  AND  DISEASE;  GENERAL  CONSIDERATIONS..   11(> 

Koch's  Rules,  116. — Animal  Inoculation,  117. — Interrelationships  of  the  Organ- 
i-ni  and  the  Body,  119. 

CHAPTER  XIII.— IMMUNITY.     GENERAL  DISCUSSION 121 

Immunity,  121. — External  Resistance  to  Infection,  121. — Variations  of  Individu- 
als in  Susceptibility  to  Disease.  Pri-di-posing  Factors,  122. — Types  of  Immun- 

122. — Natural  Immunity,  122. — Acquired  Immunity,  123. — Active  Ac-quin-d 
Immunity,  123. — Acquired  Passive  Immunity,  125.— Tli«>ri<-s<,t  Immunity,  12.r>. 
— Theory  of  Exhaustion.  125. — Noxious  Ret'cntion  Thc-ory,  126. — MetchnikofT'.s 

>rv  of  Phagocytosis,  126.— Ehrlich'M  Humoral  Theory.  120.— Duration  of 
Immunity,  127. — Antigens  and  Antibodies,  IL'7.  -Antibodies  HM  F;u-tors  in  Ac- 
quired Immunity,  127. 


CONTENTS  13 

PAGE 

CHAPTER  XIV.— ANTITOXINS  AND  RELATED  ANTIBODIES .129 

Antibodies  of  Ehrlich's  First  Order,  129. — Toxins,  129. — Antitoxins,  131. — Con- 
stitution of  the  Toxin,  134. — Constitution  of  Antitoxin,  134. — Diagrammatic 
Representation  of  Toxin  and  Antitoxins,  135. — Preferential  Union  of  Toxins  with 
Body-cells,  135. — Antitoxins  of  Commercial  Importance,  136. — Manufacture  of 
Diphtheria  Toxin  and  Antitoxin,  136. — Preparation  of  Tetanus  Toxin  and  Anti- 
toxin, 143. — Preparation  of  Other  Toxins  and  Antitoxins,  145. — Antienzymes,  145. 
— Other  Antibodies  Related  to  Antitoxins,  146. 

CHAPTER  XV. — AGGLUTINATION  AND  PRECIPITATION 147 

Antibodies  of  Ehrlich's  Second  Order,  147. — Differentiation  of  Precipitation  and 
Agglutination,  147. — Agglutination,  147. — Precipitins,  154. 

CHAPTER  XVI. — CYTOLYSINS,  INCLUDING  BACTERIOLYSINS,  AND  HEMO- 

LYSINS 157 

Antibodies  of  Ehrlich's  Third  Order,  157. — Cytolysins,  157. — Group  Cytolysins, 
160. — Bacteriolysins,  160. — Hemolysins,  162. — Fixation  of  Complement  and  Its 
Utilization,  163. — Cytotoxins,  165. 

CHAPTER  XVII. — OPSONINS  AND  PHAGOCYTOSIS 166 

Opsonins,  167. — Opsonic  Index,  169. — Autogenic  Vaccines,  173. — Passive 
Opsonic  Immunization,  175. 

CHAPTER  XVIII. — ANAPHYLAXIS  AND  HYPERSUSCEPTIBILITY 176 

Phenomenon  of  Arthus,  176.— Serum  Sickness  in  Man,  176.— Theobald  Smith 
Phenomenon,  177. — Antibodies  in  Anaphylaxis,  177. — Relationship  of  Ana- 
phylaxis  to  Certain  Body  Reactions,  179. 

CHAPTER  XIX. — AGGRESSINS.  .  181 


SECTION  IV 

PATHOGENIC  MICROORGANISMS  EXCLUSIVE  OF  THE  PROTOZOA 
CHAPTER  XX. — MICROORGANISMS  AS  A  CAUSE  OF  DISEASE 183 

Infectious  Diseases,  183. — Contagious  Diseases,  183. — Bacteria  Normally  Present 
in  the  Body  or  on  its  Surface,  184. — Types  of  Disease  Produced  by  Microorgan- 
isms, 186.— How  Bacteria  Produce  Disease,  187. — Groups  of  Pathogenic  Micro- 
organisms Exclusive  of  Protozoa,  187. 

CHAPTER  XXI. — NON-SPECIFIC  PYOGENIC  Cocci 190 

Micrococcus  Aureus,  192. — Micrococcus  Albus,  196. — Micrococcus  Citreus, 
196. — Micrococci  of  Uncertain  Significance,  196. — Streptococcus  Pyogenes,  197. — 
Streptococcus  Lacticus,  204. 

CHAPTER  XXII. — SPECIFIC  INFECTIOUS  DISEASES  PRODUCED  BY  Cocci.  .  207 

Streptococcus  Equi,  207. — Streptococcus  Gallinarum,  210. — Streptococcus  Sp., 
211. — Streptococcus  Sp.,  213. — Other  Streptococci  of  Uncertain  Significance, 
214. — Streptococcus  Pneumonias,  215. — Micrococcus  Meningitidis,  218. — Micro- 
coccus  Intracellularis  Equi,  220. — Micrococcus  Melitensis,  221.— Micrococcus 
Caprinus,  222. — Micrococcus  Gonorrhoeae,  224. — Micrococcus  Ascoformans,  226. 

CHAPTER  XXIII. — NON-SPECIFIC  PYOGENIC  BACILLI 228 

Bacillus  Pyocyaneus,  228. — Bacillus  Pyogenes  Suis,  230. 

CHAPTER  XXIV. — DIPHTHERIA  GROUP 231 

Bacillus  Diphtheria,  231.— Bacillus  Pseudodiphthericus,  236. 


14  CONTEXTS 

PAGE 

CHAPTER  XXV.  —  BACILLUS  PSEUDO-TUBERCULOSIS  GROUP  ............   238 

Bacillus  Pseudotuberculosis.  238.  —  Bacillus  Lymphangitidis  I'lcprosa,  241.  — 
Bacillus  Pyogenes  Bovis,  242 

CHAPTER  XXVI.  —  SWINE  ERYSIPELAS  GROUP  .......................   244 

Bacillus  Rhusiopathise,  244.  —  Bacillus  Murisepticus,  248. 

CHAPTKK  XXVII.—  GLANDERS  GROUP  ..............................  250 

Bacillus  Mallei,  250.—  Bacilli  of  Selter,  Babes,  and  Kutscher,  200. 

CHAPTER  XXVIII.  —  INTESTINAL  OR   COLON-TYPHOID   GROUP.     WATER 

ANALYSIS  ................................................  261 

Subgroup  I—  Colo,,  Suh^ro,,,,.  202.  —Bacillus  Coli,  262.—  Bacillus  Lactis  Aerog- 
enes,  266.  —  Bacillus  Pneumoniae,  267.  —  Subgroup  II  —  Intermediate,  Hog-cholera, 
or  Enteritidis  Subgroup,  268.  —  Bacillus  Enteritidis,  268.  —  Bacillus  Cholera?  Suis, 
271.  —  Bacillus  Paratyphosus,  274.  —  Bacillus  Psittacosis,  276.  —  Bacillus  Typhi 
Murium,  276.  —  Bacillus  Pullorum,  277.  —  Subgroup  III  —  Typhoid-dysentery 
Subgroup,  278.—  Bacillus  Typhosus,  278.—  Bacillus  Dysenterise,  282.—  Bacteria 
of  Water  and  Water  Purification,  284.  —  Quantitative  Examination  of  Water,  285.  — 
Qualitative  Examination  of  Water,  287.  —  Water  Purification,  289. 

CHAPTER  XXIX.—  HEMORRHAGIC  SEPTICEMIA  GROUP  ................   293 

Bacillus  Avisepticus,  295.  —  Bacillus  Suisepticus,  298.  —  Bacillus  Boyisepticus, 
302.  —  Other  Hemorrhagic  Septicemias  of  Animals,  303.  —  Bacillus  Pestis,  303. 

CHAPTER  XXX.  —  ACID-FAST  GROUP  ................................   30S 

Bacillus  Tuberculosis,  308.—  Bacillus  of  Johnes'  Disease,  326.—  Bacillus  Leprae, 
328.  —  Non-pathogenic  Acid-fast  Bacteria,  .'{29. 

CHAPTER  XXXI.  —  ANTHRAX  GROUP  ...............................   331 

Bacillus  Anthracis,  331.  —  Bacillus  Lactimorbi,  33S. 

CHAPTER  XXXII.  —  ABORTION  BACILLI  s  (  IHOUP  .....................   341 

Bacillus  Abort  u>.  311. 

CHAPTER  XXXIII.  —  BACILLUS  NECROPHORUS  GROUP  ................   345 

Bacillus  Necrophoru.-,  :U">. 

CHAPTER  XXXIV.  —  GROUP  OF  SPORE-BEARING  ANAEROBES  ..........  349 

Bacillus  Tetani,  349.  —  Bacillus  Chauvsei,  355.  —  Bacillus  Gastromycosis  Ovis,  359. 
—  Bacillus  (Edematis,  359.—  Bacillus  Wclchii,  361.  —  Bacillus  Botulinu*,  364. 

CHAPTKK  XXXV.—  Vnmio  OR  CHOLERA  SPIRILLUM  C.m.ri'  ...........   367 

Spirillum    Meichnikovi.  .'<i>7.      Spirillum  Cholera^,  369.  —  Non-pathogenic  Spirilla, 

CHAPTER  XXXVI.  —  ACTINOMYCES  GROUP  ..........................   372 

momycea  Bovis,  375.  —  Actinomyces  \ocanlii,  :',7v  Actinomyccs  Caprse, 
380.  —  Actinomyces  Madura?,  381.  —  ActinomyCM  llppingeri,  382.  —  Actinomyces 
of  Other  Ir.fertioMs  382. 

ClIAPlll;     .\\.\\II.         Bl.\ST<>  \nrKTKS  .............................         383 

Blaatomycefl    Farcimiii..-n-,    :;M 
myccs  CoeekHoUes,  :'^'» 

ClI  \I-TI-.K     \\.\\III.—  MOLD    OH     1  I  YPIIMM  \«'I.TK    C.uoll'  ..............     392 

The  Genut   Anprr(jillit.".    W2.    -Aspergillu>    I  uimi.'atii>,   :{!i.",.      Asj»ergillus 
'  .      Ot 


398.  —  AflpergUrai     N'urer.    :<!»'.».      Other    S)M-ci.-s    ,,f     \^p,-rgilli.     MKt.      T)»-    Cenus 
Pen  I  nil,  urn.    \  <«).  —  7'/,,     <,,„„     FuMtHum,     I'll         I  ii-:irium    Ivniinuni,  402.  —  The 
,      Sjiurnini-lnnii.    tUl.      Sporof  richiim    Hem  m.-nmi.    I'll.       '/'/,,    (i.mrn    Tricho- 
/<//v/"»  <ii"       '  on,  .\<  >uir,  HI,,  tun!  ftiiliiiiii.    JDS.      Trichophx  ton  Tonsiirans, 

ins       Afhonon  Schoenl-inn,  -»«i«i.      (  Mdiiiin  A  ll.i>  •:.  r.-.    IHl. 


CONTEXTS  15 

SECTION  V 

PATHOGENIC  PROTOZOA 

PAGE 

CHAPTER  XXXIX. — STRUCTURE,  RELATIONSHIPS,  AND  CLASSIFICATION- 

OF  THE  PROTOZOA 412 

Structure  of  the  Protozoa,  412.— Classification  of  the  Protozoa,  414. 

CHAPTER  XL.— PATHOGENIC    PROTOZOA    OF    THE    CLASS    SARCODIXA 

(RHIZOPODA) 415 

The  Genus  Entamaeba,  416. — Staining  Methods,  417. — Methods  of  Isolation 
and  Cultivation,  417. — Entanufiba  Coli,  418. — Entamceba  Histolytica,  419. — Ent- 
amoeba  Tetragena,  422. 

CHAPTER  XLI. — PATHOGENIC  PROTOZOA  OF  THE  MASTIGOPHORA   (EX- 
CLUSIVE OF  THE  SPIROCHETES) 423 

The  Genus  Trypanosoma,  423. — Morphology,  424. — Cultivation  of  Trypanosomes, 
426. — Method  of  Disease  Production,  426. — Examination  and  Staining  Methods, 
426. — Trypanosoma  Equiperdum,  426. — Trypanosoma  Evansi,  428. — Trypano- 
soma Brucei,  429. — Trypanosoma  Equinum.  431. — Trypanosoma  Dimorphon,  432 
— Trypanosoma  Congolense,  433. — Trypanosoma  Pecaudi,  433. — Trypanosoma 
Cazalboui,  434. — Trypanosoma  Theileri,  435. — Trypanosoma  Gambiense,  436. — 
Trypanosoma  Cruzi,  437. — Trypanosoma  Calmettei,  437. — Trypanosomes  in 
Birds,  437. — Trypanosoma  Lewisi,  437. — The  Genus  Herpetomonas,  438. — Herpet- 
omonas  Donovani,  438. — Leishmannia  (Herpetomonas?)  Infantum,  439. — 
Leishmannia  Tropica,  439. 

CHAPTER  XLII. — SPIROCHETE  GROUP 440 

Spirochseta  Obermeieri,  444. — Spirochaeta  Duttoni,  446. — Spirochaeta  Kochi, 
447. — Spirochseta  Anserina  or  Gallinarum,  448. — Spirochaeta  Theileri,  450. — 
Spirochaeta  Pallida,  451. — Spirochaeta  Pertenuis,  454. — Other  Spirochetes,  454. 

CHAPTER  XLIII. — SPOROZOA 455 

The  Genus  Piroplasma  or  Babesia,  456. — Piroplasma  Bigeminum,  456. — Piro- 
plasma  Parvum,  458. — Piroplasma  Mutans,  458. — Piroplasma  Equi,  458. — 
Piroplasma  Ovis,  460. — Piroplasma  Canis,  461. — Piroplasma  Gibsoni,  462. — 
Piroplasma  Commune,  462. — The  Genus  Plasmodium,  462. — Plasmodium  Vivax, 
463. — Plasmodium  Malarise,  464. — Plasmodium  Immaculatum  and  Falciparum, 
465. — The  Genera  Proteosoma,  Halteridium,  and  Hemoproteus,  465. — The  Genus 
Anaplasma,  465. — Anaplasma  Marginale,  465. — The  Genus  Leucocytozobn,  467.— 
The  Genus  Sarcocystis,  468.— The  Genus  Coccidium,  469. — Coccidium  Tenellum, 
470. — Coccidium  Cuniculi,  472. — Coccidium  of  Cattle,  473. — Coccidium  of 
Sheep.  473. 

CHAPTER  XLIV. — PATHOGENIC  PROTOZOA  OF  THE  INFUSORIA 474 

Balantidium  Coli,  474. 

SECTION  VI 

INFECTIOUS  DISEASES  IN  WHICH  THE  SPECIFIC  CAUSE  IS  NOT 
CERTAINLY  KNOWN 

CHAPTER  XLY. — DISEASES  PRODUCED  BY  ULTRAMICROSCOPIC  ORGAN- 
ISMS    476 

Bacterial  or  Protozoan  Relationships  of  Ultramicroscopic  Organisms,  477. — 
Virus  of  Pleuropneumonia,  478. — Virus  of  Foot-and-mouth  Disease,  479. — 
Virus  of  Rinderpest  or  Cattle  Plague,  480. — Virus  of  Hog-cholera,  481. — Virus  of 
Horse  Sickness,  483. — Virus  of  Infectious  Anemia  of  the  Horse,  484. — Virus  of 
Dog  Distemper,  485. — Virus  of  Fowl  Plague,  486. — Virus  of  Epithelioma  Con- 
tagiosum,  487. — Virus  of  the  Poxes,  488.— Virus  of  Yellow  Fever,  488. — Virus 
of  Epidemic  Infantile  Paralysis,  488. — Virus  of  Rabies,  489. 

BIBLIOGRAPHICAL  INDEX 493 

INDEX..  497 


VETERINARY  BACTERIOLOGY 


SECTION    I 

MORPHOLOGY,  PHYSIOLOGY,   AND   CLASSIFICATION 
OF    BACTERIA 


CHAPTER  I 

INTRODUCTION 

BACTERIOLOGY  may  be  defined  as  that  branch  of  science  which 
treats  of  bacteria,  their  forms,  and  functions.  Since  the  true 
bacteria  are  plants,  this  science  may  be  considered  as  a  sub- 
division of  the  great  mother  science  of  botany.  Bacteria  are 
the  direct  or  indirect  causes  of  many  pathologic  conditions  in 
the  bodies  of  animals,  their  study  may  accordingly  be  regarded  also 
as  one  of  the  fundamental  sciences  underlying  medicine. 

Certain  of  the  microscopic  unicellular  animals  (protozoa)  are 
likewise  commonly  considered  in  a  discussion  of  bacteriology, 
for  some  of  them  are  known  to  cause  disease,  and  have  been 
studied  largely  by  means  of  the  laboratory  technic  developed 
by  the  bacteriologist.  There  are  several  other  reasons  for  this 
inclusion:  the  dividing  line  between  bacteria  and  protozoa  is  far 
from  distinct,  and  it  is  impossible  to  determine  in  many  cases 
from  a  superficial  examination  of  a  new  disease  whether  it  is 
caused  by  the  invasion  of  true  bacteria  or  protozoa.  Further- 
more, the  line  of  demarcation  between  the  true  bacteria  and  that 
group  of  plants  known  as  fungi  (including  molds,  mildews,  smuts, 
rusts,  toadstools,  puff-balls,  etc.)  is  very  poorly  defined.  Several 
of  the  molds  and  yeasts  are  disease  producing,  and  are  generally 
included  in  a  discussion  of  bacteriology.  Certain  worms,  mites, 


18  VETERINARY  BACTERIOLOGY 

insects,  and  other  higher  forms  of  animal  life  are  likewise  fre- 
quently parasitic,  but  are  excluded  from  consideration  here. 

Medical  bacteriology  may  be  defined  as  that  branch  of  bacteri- 
ology which  treats  of  those  microorganisms  (including  the  true  bac- 
teria, molds,  and  protozoa)  that  produce  disease  in  the  animal 
body  or  are  related  directly  or  indirectly  to  the  maintenance  of 
health. 

The  history  of  veterinary  bacteriology  is  closely  linked  with  that 
of  general  medical  bacteriology,  for  many  of  the  diseases  of  man 
are  transmissible  to  animals  and  vice  versa.  It  should  be  remem- 
bered that  both  are  merely  subdivisions  of  a  great  science,  concern- 
ing which  it  is  important  that  the  student  should  gain  some- 
thing of  a  perspective  view,  particularly  with  reference  to  its 
history  and  development.  This  development  has  been  so  rapid, 


Fig.  1. — Leeuwenhoek's  drawings  of  bacteria:  A,  B,  Bacilli,  probably;  C-D, 
path  of  movement;  E,  cocci;  F,  Leptothrix,  probably;  G,  spirillum. 

and  so  many  of  the  important  discoveries  have  been  made  within 
recent  years,  that  it  is  frequently  difficult  to  determine  their 
relative  importance.  However,  certain  facts  and  personalities 
stand  out  so  conspicuously  that  they  are  deserving  of  brief  con- 
sideration. 

The  Microscope  and  its  Influence. — The  existence  of  living 
plants  or  animals  smaller  than  can  be  seen  by  the  unaided  eye 
was  conjectured  by  several  of  the  Greek  philosophers  and  phy- 
sicians who  used  such  theories  in  their  speculations  on  the  origin 
and  cause  of  fermentation  and  disease.  Until  the  discovery 
of  the  microscope  such  speculations  were  without  any  basis  in 
fact. 

Lccmvcnhook  (1632-1723),  in  the  course  of  his  examinations 
of  a  great  variety  of  natural  objects  by  means  of  the  somewhat 


INTRODUCTION  19 

crude  lenses  of  his  own  manufacture,  chanced  to  observe  the 
presence  of  motile  and  motionless  microorganisms  in  the  tartar 
from  teeth  and  in  various  decaying  organic  materials.  His 
correspondence  with  the  Royal  Society  of  London  and  the  figures 
published  leave  no  doubt  but  that  he  actually  observed  bacteria. 
These  drawings  are  of  such  historic  interest  that  they  are  here 
reproduced. 

Each  advance  in  the  efficiency  of  the  microscope  was  followed 
by  an  advance  in  our  knowledge  of  the  microorganisms,  although 
speculation  frequently  outran  the  ability  to  see  clearly.  The  com- 
pound microscope  has  proved  to  be  indispensable  in  the  study  of 
these  forms.  Since  the  introduction  of  this  instrument  the  degree 
of  magnification,  the  clearness  of  definition,  and  the  mechanic  ar- 
rangements for  accurate  focusing  have  been  gradually  improved 
until  at  the  present  time  the  homogeneous  oil  immersion  objective, 
the  compensating  ocular,  and  the  Abbe  condenser  are  in  constant 
use  in  the  laboratory,  and  enable  us  to  secure  readily  magnifica- 
tion to  1500  diameters  or  more.  During  the  last  several  decades 
there  has  been  little  increase  in  magnification,  due  to  two  reasons. 
The  greater  the  magnification  the  more  convex  and  consequently 
the  smaller  must  be  the  lenses  used  in  the  objectives,  and  the  more 
difficult  becomes  their  grinding  and  adjustment.  Furthermore, 
the  physicist  tells  us  that  a  clear  view,  with  determination  of  the 
size  and  shape  of  microscopic  objects,  cannot  be  obtained  when  the 
objects  examined  are  smaller  than  one-half  the  wave  length 
of  the  rays  of  light  in  which  they  are  examined.  There  is  thus 
a  seemingly  insurmountable  barrier  set  to  an  indefinite  increase 
in  magnification. 

A  recent  advance  has  been  made  through  the  development  of 
the  ultramicroscope.  This  has  made  visible  objects  much  smaller 
than  those  which  had  been  previously  observed.  A  bright  gleam 
of  light  from  an  arc  or  similar  source  is  passed  across  the  darkened 
field  of  the  microscope,  and  the  light  is  reflected  to  the  eye  from 
any  particles  that  may  be  in  suspension.  These  objects  are 
seen  in  the  same  manner  that  minute  particles  of  dust  are  made 
visible  in  a  bright  ray  of  light  that  enters  a  darkened  room.  The 
use  of  the  ultramicroscope  has  not  as  yet  added  many  facts  of 
value  to  our  knowledge  of  the  bacteria. 


20  VETERINARY   BACTERIOLOGY 

Nature  and  Classification  of  Microorganisms. — Leeuwenhoek, 
whom  we  have  seen  to  be  the  first  observer  of  bacteria,  contributed 
very  little  to  a  knowledge  of  their  essential  nature.  F.  Muller 
(1786)  worked  out  a  simple  classification,  but  did  not  differentiate 
between  bacteria  and  protozoa.  To  him  we  owe  several  of  the 
group  names  applied  to  bacteria,  such  as  bacillus,  vibrio,  spirillum. 
Ehrenberg  (1795-1875),  with  the  improved  microscope  and 
lenses  at  his  command,  prepared  the  first  logical  classification  of 
bacteria.  He  differentiated  the  true  bacteria  from  the  protozoa, 
and  his  arrangement  is  the  basis  for  the  classification  used  most 
extensively  at  present.  Cohn  (1828-1898)  elaborated  and  modi- 
fied Ehrenberg's  classification.  With  the  continued  improvement 
in  the  microscope  and  laboratory  technic,  more  careful  studies 
of  structure,  form,  and  relationships  have  been  rendered  pos- 
sible, and  many  classifications  and  groupings  for  bacteria 
have  been  suggested.  The  difficulty  in  finding  morphologic 
characters  that  are  accurate  indices  to  true  relationships 
has  made  the  subject  a  troublesome  one.  The  classification 
of  bacteria  most  commonly  in  use  at  present  is  that  of  Migula, 
published  in  1900  in  Engler  and  Prantl's  "  Synopsis  of  the  Genera 
of  Plants."  As  will  be  seen  from  the  discussion  recorded  in 
Chapter  V,  under  the  heading  of  Classification,  even  this  system 
is  not  wholly  satisfactory. 

Spontaneous  Generation. — In  ancient  times  and  even  during 
the  middle  ages  it  was  generally  held  by  the  philosophers  and 
scientists  that  living  things,  animals,  and  plants,  could  arise  de 
novo.  Among  the  first  observations  that  created  doubt  in  man's 
mind  as  to  the  validity  of  this  belief  was  that  of  Francisco  Redi, 
who  covered  meat  with  gauze  to  protect  it  from  flies,  and  found  that 
maggots  did  not  develop  in  it  spontaneously,  but  arose  from  the 
eggs  which  the  flies  deposited  on  the  screen.  This  pointed  the 
path  for  other  similar  studies,  and  it  was  not  long  before  the  idea 
of  spontaneous  generation  of  the  higher  forms  of  life  was  aban- 
doned. When  the  microscope  revealed  the  presence  of  myriads  <>l 
microorganisms  in  all  decaying  or  putrefying  materials,  it  was 
concluded  that  these  organisms  arose  without  progenitors  of  their 
own  kind,  hut  directly  from  the  organic  materials  of  their  sur- 
roundings. Boiling  was  believed  to  certainly  destroy  all  life. 


INTRODUCTION  21 

yet  it  was  found  that  boiled  decoctions  would  not  always  remain 
free  from  microorganisms.  The  theory  of  spontaneous  generation 
of  these  bacteria  was  opposed  by  some  and  supported  vigorously  by 
others  of  the  best  scientists  of  the  time.  Experiments  were  care- 
fully planned  and  a  great  variety  of  materials  used,  paving  the  way 
for  the  development  later  of  the  laboratory  technic  of  the  bacteriol- 
ogist. The  sterilizing  action  of  heat,  the  antiseptic  action  of 
certain  chemicals,  and  the  value  of  the  cotton  plug  as  a  bacterial 
filter  were  demonstrated.  The  theory  of  spontaneous  generation 
as  a  topic  of  contention  practically  disappeared  about  1860. 
This  was  largely  due  to  the  efforts  of  Pasteur,  who,  by  a  long 
series  of  ingenious  experiments,  overthrew  the  last  defense  of  the 
supporters  of  the  theory.  The  dictum,  omne  viuum  ex  vivo  (all 
life  from  life),  is  universally  accepted  at  the  present  time,  and  the 
controversy  has  little  but  historic  interest. 

Relationship  of  Microorganisms  to  Fermentation  and  Decay.— 
As  has  been  previously  noted,  some  of  the  early  philosophers 
hazarded  the  opinion  that  decay  might  be  caused  by  invisible 
living  beings  of  some  kind.  The  causal  relationship  of  micro- 
organisms to  decay  and  particularly  to  fermentation  was  first 
definitely  established  by  the  work  of  Louis  Pasteur  (1822-1895). 
He  found  the  production  of  alcohol  and  carbon  dioxid  from  sugar 
was  due  to  a  yeast,  that  milk  soured  because  of  the  activity  of 
bacteria,  and  that  many  of  the  familiar  changes  in  organic  sub- 
stances were  accomplished  by  microorganisms.  His  conclusions 
were  strenuously  opposed  and  ridiculed  by  the  great  German 
chemist,  Liebig.  Doubtless  the  necessity  for  meeting  the  attacks 
of  the  latter  and  of  establishing  his  points  beyond  possibility 
of  refutation  led  him  to  devise  and  develop  many  of  the  laboratory 
methods  in  common  use  at  the  present  time.  As  a  result  of 
Pasteur's  work  the  fundamental  importance  of  bacteria  in  the  trans- 
formation of  nitrogen  and  carbon  compounds  in  nature,  the 
disposal  of  waste,  the  purification  of  water,  the  enriching  of  the 
soil,  and  many  of  the  changes  in  the  manufacture  of  foods  have 
been  established. 

Relationship  of  Microorganisms  to  Disease. — The  probable 
causal  relationship  of  microorganisms  of  some  kind  to  disease 
was  argued  as  long  ago  as  1762  by  Blencig,  of  Vienna.  His 


22  VETERINARY  BACTERIOLOGY 

theories  were  not  generally  accepted,  and  it  was  not  until  1840 
that  Henle  proposed  what  we  have  come  to  call  the  germ  theory 
of  disease.  He  never  succeeded  in  proving  his  point  satisfactorily 
because  of  the  lack  of  proper  methods  and  technic.  Many 
other  writers  within  the  next  few  years  discussed  the  theory 
and  numerous  facts  were  adduced  in  favor  of  it.  The  majority 
of  medical  practitioners,  however,  put  very  little  faith  in  it.  The 
argument  that  certain  organisms  were  always  present  was  met 
with  the  statement  that  these  organisms  were  the  result  and  not 
the  cause  of  the  disease.  Davaine  (1863)  practically  demon- 
strated by  inoculation  experiments  the  causal  relationship  of  a 
bacillus  he  found  in  the  blood  of  diseased  animals  to  anthrax. 
Pasteur  (1865)  proved  the  cause  of  a  silkworm  disease  to  be  a 
protozoan  parasite.  Koch  and  Pasteur  later  cultivated  the 
anthrax  organism  in  the  laboratory  and  showed  beyond  a  doubt 
its  relationship  to  the  specific  disease.  Improved  laboratory 
technic  cleared  up  the  cause  of  many  diseases  within  the  next  de- 
cade or  two.  The  discovery  of  the  Bacillus  tuberculosis  by  Koch 
(1882)  marks  the  real  beginning  of  bacteriologic  science.  The 
knowledge  of  protozoa  as  a  cause  of  disease  lagged  somewhat 
behind  that  of  bacterial  infections.  Evans  (1880)  described 
the  trypanosome  of  surra  and  transmitted  the  disease  by  inocula- 
tion experiments.  In  1882  Laveran  observed  the  Plasmodium 
malarice,  the  cause  of  malaria. 

Development  of  Laboratory  Methods. — Progress  was  delayed 
in  the  study  of  objects  as  minute  as  the  bacteria  because  of  the 
lack  of  proper  methods  for  their  isolation,  observation,  and  identi- 
fication. Culture-media  in  which  the  pathogenic  microorganisms 
could  be  grown  were  used  by  Pasteur  and  Koch.  To  the  latter 
we  are  indebted  (1882)  for  our  knowledge  of  the  solid  media  which 
can  be  used  for  the  isolation  of  organisms  from  mixed  cultures. 
The  importance  of  this  contribution  can  hardly  be  overestimated, 
for  the  use  of  pure  cultures  lies  at  the  very  foundation  of  all 
modern  bacteriologic  investigation.  This  one  discovery  accounts 
in  large  measure  for  the  rapid  advance  made  during  the  next 
two  decades  in  the  identification  of  the  organisms  producing 
disease.  The  use  of  aniline  dyes  in  rendering  cells  and  their  struc- 
tun  more  plainly  visible  under  the  microscope  we  owe  to  Weigert, 


INTRODUCTION  23 

but  the  application  to  bacteriology  was  made  by  Koch.  Since 
their  introduction  successive  advances  in  staining  technic  have  in 
every  instance  been  followed  by  the  discovery  of  new  organisms 
related  to  disease.  The  microscope,  liquefiable  media,  and 
anilin  dyes  constitute  the  trio  of  most  important  factors  in  the 
development  of  the  science  of  bacteriology. 

Development  of  Theories  of  Immunity. — Knowledge  that  one 
attack  of  certain  diseases  generally  prevented  a  recurrence  and 
that  diseases  could  not  be  indiscriminately  transferred  to  all 
species  of  animals  has  existed  ever  since  the  foundation  of  medi- 
cine. Many  theories  have  been  advanced  to  account  for  this 
phenomenon.  A  few  of  the  names  of  investigators  who  have  put 
the  facts  into  logical  form  for  presentation  and  study  should 
be  considered.  Metchnikoff  conceived  that  the  white  blood-cor- 
puscles and  some  other  body  cells  acted  as  scavengers  and  des- 
troyed microorganisms  in  the  blood.  This  theory  in  modified 
form  furnishes  to-day  the  logical  basis  for  many  of  the  operations 
of  the  practitioner.  Von  Behring  (1890)  published  results  of  stud- 
ies on  the  diphtheria  bacillus  in  which  he  recounted  the  discovery 
of  the  specific  toxin  of  this  organism  and  a  specific  antitoxin. 
He  laid  the  foundation  for  the  present-day  "  humoral  "  theory 
of  immunity,  which  supplements  so  well  the  phagocytic  theories 
of  Metchnikoff.  Ehrlich  has  correlated  and  coordinated  the  vari- 
ous facts  of  the  humoral  theory  and  has  made  substantial  additions 
to  it.  He  has  created  a  terminology  which  has  been  quite  generally 
accepted  and  has  proved  most  useful  in  discussion  of  the  sub- 
ject. So  extensive  have  been  researches  in  the  field  of  immunity 
during  the  last  two  decades  (since  1890)  that  it  has  assumed 
almost  the  rank  of  a  coordinate  science. 

Development  of  Sanitary  Science  and  Preventive  Medicine.— 
In  1858  Murchison  formulated  the  so-called  pythogenic  theory  of 
disease — i.e.,  that  disease  is  caused  by  the  emanations  arising  from 
decaying  or  putrefying  organic  matter,  and  by  the  consumption 
of  such  materials  in  water  and  food.  This  theory  was  quite  com- 
monly accepted,  and  its  practical  applications  form  the  basis  for 
our  modern  sanitary  science.  The  disposal  of  sewage  and  refuse, 
the  purification  of  drinking-water,  and  adequate  systems  of  plumb- 
ing were  advocated  and  adopted  before  the  germ  theory  of  disease 


24  VETERINARY   BACTERIOLOGY 

had  been  well  formulated  and  established.  The  rapid  accumula- 
tion of  evidence  in  favor  of  the  germ  theory  gave  rise  to  the  art 
of  preventive  medicine.  Lister  (1875)  advocated  the  use  of 
antiseptics  in  the  treatment  of  wounds  and  wound  infection  as  a 
direct  method  of  combating  the  activity  of  undesirable  bacteria. 
The  discovery  of  the  means  of  transmission  in  most  of  the  infectious 
diseases  has  enabled  man  to  take  measures  for  their  eradication. 
Yellow  fever,  malaria,  diphtheria,  bubonic  plague,  and  many  other 
diseases  are  now  kept  in  check  by  the  use  of  preventive  measures 
indicated  by  our  knowledge  of  the  manner  in  which  they  spread. 
It  is  probable  that  greater  advance  will  be  made  within  the  next 
few  years  in  the  domain  of  preventive  medicine,  for  mankind 
is  fast  coming  to  realize  that  prevention  is  better  than  cure. 


CHAPTER  II 

MORPHOLOGY  AND  RELATIONSHIPS  OF  MICROORGANISMS 
CONCERNED  IN  DISEASE  PRODUCTION 

POSITION  OF  PATHOGENIC  MICROORGANISMS 

Differentiation  of  Animals  and  Plants. — The  distinctions  we 
commonly  recognize  as  differentiating  plants  from  animals  in 
large  measure  disappear  among  the  microscopic  forms  of  life. 
It  is  worth  while,  therefore,  to  discuss  the  factors  that  are  taken 
into  consideration  in  the  assignment  of  a  particular  microorgan- 
ism to  the  animal  or  the  plant  kingdom.  The  difficulty  arises 
particularly  in  the  differentiation  of  the  bacteria  and  the  protozoa. 
Bacteria,  as  will  be  shown  later,  probably  have  in  all  cases  a  cell- 
wall  which  in  function  is  closely  related  to  that  of  plants.  A 
cell-wall  is  frequently  absent  in  protozoa,  the  limiting  mem- 
brane usually  consisting  of  the  ectoplast  (see  below)  alone.  The 
composition  of  the  cell-wall  is  in  some  cases  like  that  of  many 
animals  (chitin),  in  others  like  plants  (cellulose).  In  shape 
and  habit  of  growth  and  reproduction  the  bacteria  resemble 
very  closely  certain  undoubted  plants  among  the  blue-green 
algae  and  the  mold  fungi  much  more  than  they  do  animal  forms. 
On  the  other  hand,  there  are  some  types  which  intergrade  with  the 
protozoa,  so  that  there  is  at  present  doubt  as  to  their  correct 
systematic  position.  In  the  matter  of  food  supply  and  food 
utilization  some  bacteria  resemble  higher  plants,  others  resemble 
animals.  The  possession  of  organs  of  motion  by  bacteria  has 
no  direct  bearing  on  the  subject,  inasmuch  as  undoubted  plants 
of  the  group  of  algae  and  many  of  the  protozoa  have  them.  The 
evidence,  on  the  whole,  is  much  in  favor  of  classification  of  the 
true  bacteria  with  plants. 

Before  beginning  a  study  of  the  morphology  or  structure  of 
these  microorganisms,  their  position  and  relationships  to  the 
other  groups  of  plants  and  animals  should  be  understood.  Fol- 

25 


26  VETERINARY   BACTERIOLOGY 

lowing  is  a  discussion  of  these  various  groups,  with  particular 
emphasis  upon  the  groups  or  subgroups  of  significance  in  medical 
bacteriology. 

The  plant  kingdom  is  generally  divided  into  four  great  groups, 
the  Spermatophytes,  or  seed-bearing  plants,  the  Pteridophytes,  or 
ferns  and  fern-like  plants,  the  Bryophytes,  or  moss  plants,  and  the 
Thallophytes,  including  all  plants  low  in  the  scale  of  evolution, 
that  have  not  become  highly  differentiated  and  that  never  have 
roots,  stems,  and  leaves.  All  of  the  plant-like  organisms  to  be  con- 
sidered belong  to  this  lowest  group,  Thallophytes. 

The  animal  kingdom  may  be  divided  into  the  Metazoa,  or 
higher  differentiated  types,  made  up  of  many  cells,  and  the  Pro- 
tozoa, or  unicellular  forms.  To  the  latter  group  belong  all  the 
animal-like  organisms  to  be  considered. 

Subdivisions  of  the  Thallophytes. — Following  is  a  key  or  out- 
line of  the  principal  subgroups  of  the  Thallophyta: 

A.  Unicellular  forms,  multiplying  only  by  splitting  of  the  cells 

to  form  two  equal  daughter-cells.     Never  any  sex  cells. 

I.  Cells  containing  blue-green  coloring-matter. 

1.  Schizophycece  (Cyanophycea?  or  blue-green  algae). 

II.  Cells  not  containing  blue-green  coloring-matter. 

2.  Schizomycetes  (Bacteria). 

B.  Unicellular  or  multicellular,  multiplication  by  some  method 

other    than    simple    fission.     Frequently    sexual    repro- 
duction occurs. 

I.  Cells  containing  green  coloring-matter  (chlorophyll). 

3.  Algce  (sea-weeds,  pond  scums,  etc.). 

II.  Cells  without  green  coloring-matter  (chlorophyll). 

4.  Fungi  (yeasts,  molds,  mildews,  rusts,  smuts,  toadstools, 
puffballs,  mushrooms,  etc.). 

Of  the  last  group  (Fungi)  only  two  subgroups  are  of  especial 
pathogenic  significance  for  the  veterinarian,  the  yeasts  and  the 
molds.  These  two,  with  the  bacteria,  constitute  the  three  types 
of  microorganisms  belonging  to  the  plant  kingdom  that  contain 
forms  pathogenic  for  animals. 


MORPHOLOGY  AND  RELATIONSHIPS  OF  MICROORGANISMS    27 

MORPHOLOGY  OF  BACTERIA 

Shape  of  Bacteria. — Bacteria  may  be  classified  according  to 
shape  into  spheres,  straight  rods,  bent  or  spiral  rods,  and  filaments. 

A  spherical  form  is  called  a  coccus  (pi.  cocci).  Although  the 
coccus  is  theoretically  spherical,  there  are  many  that  appear  some- 
what flattened  or  ovoid  when  in  groups  or  chains. 


Fig.  2. — Types  of  bacteria:  Cocci,  bacilli,  and  spirilla  (Jordan). 

A  straight  rod  is  called  a  bacillus  (pi.  bacilli). 

A  curved  or  spiral  rod  is  called  a  spirillum  (pi.  spirilla). 

The  filamentous  bacteria  are  those  in  which  the  organism  is 
greatly  elongated.  No  specific  name  has  been  given  to  this  form. 
Frequently  the  filamentous  type  exhibits  branching  and  in  other 
ways  resembles  the  mycelium  of  the  higher  fungi  or  molds. 


Fig.  3. — Involution  forms  of  bacteria:  1,  Bacillus  radidcola — a,  Normal 
rods;  b,  bacteroids.  2,  Bacillus  tuberculosis.  3,  Bacillus  subtilis.  4,  Bacillus 
aceti.  5,  Bacillus  pestis.  6,  Actinomyces  sp.  7,  Spirillum  sp.  8,  Micro- 
coccus  aureus — a,  Normal  forms;  b,  involution  forms. 

When  grown  under  favorable  conditions,  as  a  result  of  the 
action  of  certain  stimulation,  many  bacteria  assume  unusual  and 
abnormal  shapes.  Cells  of  this  type  are  called  involution  forms. 
Such  cells  are  not  necessarily  incapable  of  continued  growth  and 


28  VETERINARY   BACTERIOLOGY 

reproduction  of  the  more  usual  or  normal  type  when  brought 
under  favorable  conditions.  Frequently,  however,  these  cells 
soon  die  and  can  no  longer  reproduce.  Various  bacteria  differ 
considerably  with  respect  to  the  ease  with  which  they  produce 
involution  forms. 

Grouping  of  Bacterial  Cells. — The  cells  of  bacteria  are  frequently 
surrounded  by  a  gelatinous  material  (capsule,  see  below)  which 
causes  them  to  cling  together  in  groups.  This  grouping  in  the 
various  types  is  so  constant  that  it  is  used  to  differentiate  various 
genera  from  each  other,  and  in  some  cases  the  species  within 

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Fig.  4.  —  Development  of  groups  of  cocci:  1,  Development  of  strepto- 
coccus; 2,  development  of  micrococcus;  3,  development  of  sarcina;  4, 
development  of  staphylococcus. 

the  genus.  Bacterial  cells  divide  normally  at  right  angles  to  the 
longest  axis  of  the  cell.  This  allows  of  but  little  variation  in  the 
grouping  of  elongated  types,  but  as  the  cocci  have  no  "  longest 
axis"  they  divide  in  various  planes. 

Some  cocci  divide  constantly  in  the  same  plane  or  in  a  plane 
parallel  to  the  first.  This  results  in  the  formation  of  a  chain 
of  cocci.  An  organism  showing  this  grouping  is  culled  a  strepto- 
coccus (pi.  streptococci). 

other  cocci  divide  alternately  in  two  planes  at  right  angles 
to  each  other.  Such  an  organism  will  be  found  in  twos  (diplo- 
eoccus)  or  in  fours  (tetrad  or  tetracoccus)  .  Diplococci  may  also 


MORPHOLOGY   AND   RELATIONSHIPS   OF   MICROORGANISMS      29 

be  produced  by  the  breaking  up  of  chains  of  streptococci  into 
pairs. 

Cocci  sometimes  divide  in  three  planes  at  right  angles  to  each 
other.  This  results  in  the  formation  of  cubes  or  packets  of  cocci. 
A  packet  of  this  kind  is  called  a  sarcina  (pi.  sarcince). 


0. 


Fig.  5. — Shapes  and  groups  of  cocci:  1,  Single  coccus  (micrococcus) ;  2, 
cocci  in  an  irregular  mass  (staphylococcus) ;  3,  spheric  diplococci;  4,  flat- 
tened diplococci;  5,  coffee-bean-shaped  diplococci;  6,  lanceolate  diplo- 
cocci; 7,  streptococcus  with  short  chains;  8,  streptococcus  with  long 
chains;  9,  a  streptococcus  made  up  of  diplococcus  elements;  10,  cap- 
sulated  micrococci;  11,  sarcina. 

A  staphylococcus  (pi.  staphylococci)  is  a  coccus  whose  planes 
of  division  are  not  at  right  angles,  or  which  divides  at  different 
intervals  with  a  consequent  irregular  grouping  of  the  cells  much 
resembling  grapes  in  a  cluster. 


Fig.  6.— Types  of  bacilli. 

Bacilli  occur  either  singly  or  in  chains.  The  latter  are  some- 
times known  as  streptobacilli. 

Spirilla  usually  occur  singly,  although  short  chains  of  two  or 
three  individuals  are  sometimes  observed. 

In  a  few  bacteria  the  gelatinous  envelop  of  the  cell  is  greatly 


30 


VETERINARY    BACTERIOLOGY 


thickened,  and  the  bacteria,  either  cocci  or  bacilli,  are  imbedded  in 
a  mass  of  gelatin.     Such  a  mass  of  cells  is  called  a  zooglea. 

Size  of  Bacteria. — The  unit  of  microscopic  measurement  is 
the  micron  (pi.  micra  or  microns)  and  is  indicated  by  the  Greek 


Fig.  7. — Types  of  spirilla. 

letter  p.     It  is  the  one-thousandth  part  of  a  millimeter  or,  approx- 
imately, the  one  twenty-five-thousandth  part  of  an  inch. 

Bacteria  vary  considerably  in   size,  from   forms  OJj^  or  less 
in    diameter,    barely    visible    under   the   microscope,    to   forms 


Fig.  8.— Types  of  filamentous  bacteria:  A,  Leptothrix;  B,  Cladothrix;  C, 
Nocardia;  D,  Actinomyces  or  Streptothrix. 

100  H*  or  more  in  length.  Most  bacteria  are  bet  ween  ().f>  and 
5  li  in  diameter  and  0.5  {t  and  10  ^  in  length.  Some  bacteria 
are  undoubtedly  too  small  to  be  seen  with  the  highest  power  <  of 
our  microscopes,  hence  less  than  0.1  jJ.  in  diameter.  We  know  of  the 
existence  of  these  organisms  by  their  effects.  The  organism  caus- 


MORPHOLOGY   AND    RELATIONSHIPS   OF   MICROORGANISMS      31 

ing  hog  cholera,  for  example,  is  so  small  that  it  will  pass  through 
the  pores  of  a  fine  porcelain  filter,  and  will  cause  disease  when 
injected  into  a  healthy  pig.  Such  an  organism  is  frequently  spoken 
of  as  ultramicroscopic,  or  as  a  filterable  virus. 

Histology  and  Structure  of  Bacteria. — This  topic  may  be  treated 
under  four  subheads,  the  cell  wall  with  its  related  sheaths  and  cap- 
sules, the  protoplasm,  the  cell  inclusions,  and  the  flagella. 

Cell  Wall. — The  bacterial  cell  is  in  all  cases  surrounded  by  a 
definite  membrane  that  morphologically  resembles  the  cell  wall 
of  higher  plants.  When  tested  chemically  with  various  reagents 
and  examined  microscopically,  it  is  sometimes  found  to  give 
the  reactions  characteristic  of  chitin,  the  material  which  makes 
up  the  hard  outer  shell  of  insects,  and  is  found  as  a  cell  mem- 


Fig.  9. — Capsulated  bacteria. 

brane  in  many  animals.  Chemically  chitin  is  an  ammo-sub- 
stitution product  of  a  carbohydrate.  The  fact  that  the  cell 
walls  in  bacteria  so  frequently  resemble  in  composition  those 
of  certain  animals  has  been  used  as  an  argument  for  the  animal 
relationships  of  the  bacteria.  This  is  negatived,  however,  by 
the  fact  that  in  numerous  molds  and  other  fungi,  undoubted 
plants,  the  cell  walls  are  made  up  of  a  similar  substance. 

The  cell  wall  in  bacteria  is  usually  covered  by  a  layer  of  mucil- 
aginous material,  in  most  cases  so  thin  that  the  most  careful  technic 
must  be  employed  in  its  demonstration,  in  other  cases  a  thick 
coating  or  capsule.  The  nature  of  this  capsular  substance  has 
been  a  fertile  subject  for  dispute.  A  few  bacteriologists  have 
claimed  that  it  is  composed  of  living  protoplasm,  the  majority, 


32  VETERINARY    BACTERIOLOGY 

with  seeming  justification,  believe  that  this  is  either  an  excreted 
material  or  merely  an  outer  swollen  and  differentiated  portion 
of  the  cell  wall.  Chemically  this  material  differs  in  the  various 
capsulated  bacteria.  In  some  cases  it  is  composed  of  mucin,  a 
slimy  material  made  up  of  a  protein-like  substance  united  with 
a  carbohydrate,  and  resembling  the  mucus  secreted  by  certain 
body  cells.  In  other  cases  the  capsule  is  made  up  of  pure  carbo- 
hydrates and  is  closely  allied  to  certain  of  the  vegetable  gums,  such 
as  gum  arabic  and  gum  tragacanth.  The  capsule  of  some  bacteria 
is  partially  or  wholly  soluble  in  water.  When  such  an  organ- 
ism grows  in  suitable  nutrient  solution  it  renders  the  medium 
slimy  or  gelatinous.  Slimy  milk,  bread  and  whey  are  caused  by 
the  luxuriant  growth  of  such  organisms. 


Fig.  10. — Bacteria  showing  sheaths. 

Some  of  the  filamentous  bacteria  produce  a  firm  sheath  or  tube 
outside  of  the  cell,  this  sheath  usually  inclosing  an  entire  filament 
or  chain  of  cells.  In  composition  it  probably  closely  resembles 
the  cell  wall.  In  some  cases  it  is  impregnated  with  iron  oxid, 
It  is  possible  that  the  sheath  is  a  modified  type  of  capsule. 

Cell  Protoplasm. — The  living  material  within  the  cell  wall 
is  called  the  protoplasm.  Structurally  it  may  usually  be  dif- 
ferentiated into  two  layers,  an  outer  thin  layer  lying  closely 
appressed  to  the  cell  wall,  and  an  inner  portion.  The  outer  layer 
or  ectoplast  performs  one  of  the  most  important  functions  of  the 
cell,  as  this  is  the  membrane,  and  not  the  cell  wall,  that  determines 
what  materials  in  solution  may  enter  and  what  may  leave  the 
cell;  through  it  must  pass  by  diffusion  all  the  food  that  enters  the 
cell.  When  certain  bacteria,  as  the  cholera  spirillum,  are  placed 
in  a  strong  solution  of  some  salt  which  does  not  readily  pass  through 


MORPHOLOGY    AND    RELATIONSHIPS   OF   MICROORGANISMS      33 

this  ectoplast,  the  water  from  the  cell  in  part  passes  out,  the 
protoplasm  shrinks  away  from  the  cell  wall,  and  the  cell  is  said  to 
be  plasmolyzed  (noun,  plasmolysis) .  Such  a  plasmolyzed  cell 
shows  clearly  the  ectoplast  separated  from  the  cell  wall.  When 
a  cell  of  certain  species  is  placed  in  distilled  water  or  a  concentra- 


JB 

Fig.  11. — Plasmolysis  and  plasmoptysis  of  bacterial  cells:  A,  Plasmo- 
lyzed bacterial  cells;  B,  cells  showing  plasmoptysis,  the  protoplasm  has  burst 
the  cell  wall  and  is  escaping.  (Adapted  from  Fischer.) 

tion  of  salt  considerably  less  than  that  to  which  it  has  been  accus- 
tomed, the  cell  takes  up  water,  the  cell  wall  bursts,  and  part  of  the 
protoplasm  escapes.  This  phenomenon  is  called  plasmoptysis. 
The  protoplasm  of  the  cell  is  commonly  homogeneous  in  appear- 
ance, and  stains  best  with  the  basic  aniline  dyes.  Either  a  definite 


2)  £  "  F 

Fig.  12. — Bacterial  cell  inclusions:  A,  Vacuoles  in  the  cell,  polar  staining 
rods;  B,  vacuolate  spirilla;  C,  fat  globules;  D,  glycogen  granules;  E,  meta- 
chromatic  granules;  F,  sulphur  granules. 

nucleus  is  not  present,  or  the  nuclear  material  is  so  scattered 
as  to  make  the  entire  mass  functionally  a  nucleus.  Some  bac- 
teria have  been  described  as  possessing  a  primitive  type  of 
differentiated  nucleus,  but  such  structures  cannot  be  discerned 
in  others. 

3 


34  VETERINARY   BACTERIOLOGY 

Cell  Inclusions. — Bacterial  cells  sometimes  contain  vacuoles, 
or  spaces  in  the  protoplasm  filled  with  sap  or  some  non-staining 
or  non-refractive  substance.  A  large  vacuole  near  the  center  of 
the  cell  may  crowd  the  protoplasm  to  the  ends  of  the  cell,  and 
such  organisms,  when  stained,  are  said  to  show  polar  staining. 
In  other  forms,  as  the  diphtheria  bacillus,  granules  are  formed 
that  stain  much  more  intensely  with  the  basic  aniline  dyes  than 
does  the  remainder  of  the  protoplasm.  These  are  called  meta- 
chromatic  granules.  The  function  of  these  granules  is  not  clear. 
Certain  species  of  bacteria  living  in  water  containing  hydrogen 
sulphid  are  found  to  contain  granules  of  free  sulphur  in  their 
protoplasm.  Still  others  have  food  materials  in  the  form  of  oil 
globules  or  granules  of  glycogen. 


Fig.  13. — Distribution  of  the  flagella  of  bacteria:  A,  Non-motile  or  atrich- 
ous  bacilli,  spirilla,  and  cocci;  B,  monotrichous  flagella  of  bacilli,  spirilla,  and 
cocci;  C,  lophotrichous  flagella  of  bacilli  and  spirilla;  D,  amphitrichous  flagella 
of  bacilli  and  spirilla;  E,  peritrichous  flagella  of  bacilli  and  spirilla. 

Flagella. — Many  bacteria  are  motile  by  means  of  whip-like 
threads  of  protoplasm  which  extend  from  their  surfaces.  These 
threads  are  known  as  whips  or  flagella  (sing,  flagellum).  These 
flagella  are  observed  with  difficulty  in  the  living  organism  ex- 
cept with  dark  field  illumination  and  require  peculiar  stain- 
ing technic  and  careful  treatment  to  make  them  visible  in  a 
stained  mount.  Comparatively  Jew  cocci,  many  of  the  bacilli, 
and  most  of  the  spirilla  are  flagellated.  The  distribution  of 
flagella  on  the  surface  of  the  cell  has  been  used  as  a  basis  for 
grouping.  Atrichous  bacteria  have  no  flagella;  monotrichous 
bacteria  have  a  single  flagellum  at  one  end;  lophotrichous,  a  group 
of  flagella  at  one  pole;  amphitrichous,  flagella  at  both  ends;  and 


MORPHOLOGY    AND    RELATIONSHIPS    OF    MICROORGANISMS      35 

peritrichous,  flagella  on  all  sides.  Old  cells  and  cells  transferred 
from  one  medium  to  another  are  very  apt  to  loose  their  flagella. 
A  young  culture  is  most  suitable  for  determination  of  motility. 
True  motility  must  not  be  confused  with  Brawnian  movement, 
which  is  a  vibrating  or  oscillating  motion  of  finely  divided  par- 
ticles of  almost  any  kind  suspended  in  water,  and  visible  when 
examined  under  the  microscope.  This  motion  has  not  been 
satisfactorily  explained,  but  it  is  probably  due  to  rapid  changes 
in  surface  tension  of  the  liquid  at  the  point  of  contact  with  the 
particles. 

Reproduction  in  Bacteria. — Multiplication  in  all  bacteria  is  a 
simple  process.  The  cell  commonly  elongates  or  enlarges,  a  cell 
wall  develops  across  the  middle,  and  the  two  cells  separate. 
This  operation  may  occur  with  considerable  rapidity.  Some  organ- 
isms in  favorable  medium  can  grow  to  their  full  size  and  divide  to 
form  two  individuals  in  the  course  of  twenty  minutes  to  an  hour. 
If  the  organism  could  multiply  in  this  geometric  ratio  for  a  short 
time  the  number  of  resultant  organisms  would  be  practically 
incalculable.  For  example,  if  a  bacterium  should  divide  every 
half -hour,  at  the  end  of  two  days  the  progeny  would  be  represented 
by  2s*5,  a  number  having  twenty-eight  figures.  Such  rapid  multi- 
plication is  never  long  continued,  for  food  supply  is  never  long 
favorable,  and  waste  products  of  the  bacterial  growth  tend  to 
accumulate  and  diminish  the  rate.  Nevertheless,  the  rapidity 
of  increase  of  bacteria  accounts  in  a  large  measure  for  the  consider- 
able changes  they  bring  about  in  a  short  time,  as  in  the  souring 
of  milk  or  invasion  of  the  body  in  disease. 

Many  bacteria  also  reproduce  by  means  of  spores.  These 
are  of  two  types:  endospores,  produced  inside  the  bacterial  cell, 
and  arthrospores,  consisting  of  entire  differentiated  cells.  The 
former  are  produced  by  certain  bacilli  and  spirilla,  the  latter  by 
certain  of  the  filamentous  forms  or  trichobacteria. 

Endospores  are  formed  by  many  bacilli,  and  possibly  by 
some  spirilla.  They  are  produced  in  response  to  some  definite 
stimulus,  such  as  unfavorable  conditions  of  the  environment, 
accumulation  of  waste  products,  or  change  in  reaction  of  the 
medium.  The  spore  is  essentially  a  portion  of  the  protoplasm 
of  the  cell  which  has  expelled  most. of  its  water  and  shrunken  in 


36  VETERINARY   BACTERIOLOGY 

size  until  it  occupies  a  portion  only  of  the  space  within  the  cell 
wall,  and  has  then  surrounded  itself  with  a  heavy  wall,  probably 
chitinous  in  nature.  In  practically  all  cases  there  is  but  one 
spore  in  a  cell.  The  spore  may  be  equatorial  or  polar  in  position, 
and  of  less  or  greater  diameter  than  the  cell  which  produces  it. 
The  term  dostridium  is  sometimes  used  to  indicate  a  spore-bearing 


Fig.  14. — Development  of  endospores  in  a  bacillus.     (After  Fischer.) 

rod  in  which  the  spore  is  equatorial  and  of  greater  diameter  than 
that  of  the  cell,  resulting  in  a  spindle.  Endospores  contain  only 
about  20  per  cent,  of  water  as  compared  with  80  to  90  per  cent, 
in  the  cells  which  produced  them.  An  organism  without  a  spore 
is  usually  differentiated  by  the  term  vegetative  rod  or  vegetative 
cell.  Spores  are  much  more  resistant  to  desiccation,  heat,  light, 
and  chemicals  than  the  vegetative  cells.  They  are  of  use  in 


Fig.  15. — Bacterial  spore  types:  A,  Equatorial  spores  of  a  diameter  less 
than  the  cell;  B,  polar  spores  of  a  diameter  less  than  the  cell;  C,  equatorial 
spores  of  a  diameter  greater  than  the  cell  (clostridium  type) ;  D,  drumstick  or 
polar  spores  of  a  diameter  greater  than  the  cell. 

tiding  the  organism  over  unfavorable  conditions.  Spore-bear- 
ing bacteria  are  abundant  in  the  soil,  where  they  often  are  exposed 
to  great  ranges  of  moisture,  temperature,  and  light.  When  a 
spore  again  comes  under  favorable  conditions  for  growth,  it 
germinates  and  produces  a  cell  typical  of  its  species.  Germina- 
tion is  accomplished  either  by  stretching  or  bursting  the  spore 
wall. 


MORPHOLOGY    AND    RELATIONSHIPS   OF    MICROORGANISMS      37 

Arthrospores  are  bacterial  cells  set  apart  for  purposes  of  repro- 
duction, and  are  usually  differentiated  appreciably  from  the  normal 
cell.  Several  investigators  have  claimed  that  they  are  produced 
by  some  of  the  cocci,  but  this  has  never  been  satisfactorily  estab- 


B  00  Q  fl 

y 

coo 


Fig.  16. — Germination  of  spores:  A,  Bacillus  subtilis  (Prazmowski) ;  B,  Bacil- 
lus anthracis  (deBary) ;  C,  Clostridium  sp.  (deBary) . 

lished.  The  filamentous  bacteria  or  trichobacteria  produce 
arthrospores  or  conidia,  as  they  are  sometimes  called,  by  the  dis- 
integration of  some  of  the  filaments  into  short  rods  or  spheres 
which  are  capable  of  reproducing  the  parent  type  or  by  a  process 


A  i       BC/  V)         C 

Fig.  17.  —  Arthrospores:  A,  Crenothrix  polyspora  (Cohn);  B,  GallioneHa  ferru- 
ginea,  showing  conidia  formation  (Ellis)  ;  C,  Leptothrix  ochracea  (Ellis)  . 


of  budding.  In  many  cases  the  threads  which  break  up  into 
the  spores  are  somewhat  differentiated  from  the  normal  cells  of 
the  plant,  and  are  aerial,  resembling  closely  some  of  the  molds. 


MORPHOLOGY  OF  THE  YEASTS,  SACCHAROMYCETES,  AND  BLASTO- 

MYCETES 

Yeasts,  from  the  standpoint  of  the  systematic  botanist,  are 
placed  at  some  distance  from  the  bacteria,  for  there  are  many 
differences  between  typical  yeasts  and  typical  bacteria.  On  the 
other  hand,  there  are  forms  which  intergrade  between  the  two, 


38  VETERINARY   BACTERIOLOGY 

and  are  sometimes  assigned  to  one  group,  sometimes  to  another. 
The  yeasts  and  the  molds  also  show  intermediate  types. 

Form,  Size,  and  Grouping  of  Yeasts. — Yeast  cells  are  usu- 
ally spherical,  oval,  ellipsoid,  or  cylindrical.  For  the  most  part 
they  are  larger  than  bacterial  cells,  although  there  are  excep- 
tions. The  true  yeasts  multiply  not  by  fission,  but  by  a  process 
of  budding.  The  cells  commonly  remain  united  for  a  time,  giv- 
ing rise  to  masses  consisting  of  many  individuals.  Sooner  or 
later  they  break  apart.  The  relative  shape,  size,  and  groupings 
of  the  yeast  cells  are  used  in  the  differentiation  of  species.  In 
some  species  part  of  the  cells  become  considerably  elongated  and 
form  a  kind  of  false  mycelium  resembling  that  of  the  molds. 
This  character  is  not  always  constant  in  a  given  species,  it  may 


Fig.  18. — Types  of  yeast  cells  and  groupings. 

appear  when  the  organism  is  growing  in  orte  kind  of  medium  and 
not  appear  in  another. 

Histology  and  Structure  of  the  Yeast. — The  very  young  cell 
has  no  cell  wall,  but  by  the  time  it  is  one-third  grown  the  wall 
appears  as  a  delicate  membrane.  In  old  cells  it  is  sometimes  of 
considerable  thickness.  Its  composition  has  not  been  certainly 
determined,  probably  it  is  a  carbohydrate  or  related  compound, 
and  not  chitinous,  as  are  the  walls  of  bacterial  and  mold  cells. 
To  this  substance  the  name  yeast  cellulose  has  been  given.  It 
has  not  been  prepared  free  from  nitrogen,  so  that  it  is  possible 
that  it  may  be  nitrogenous  in  nature.  It  is  sometimes  surrounded 
by  a  gelatinous  excretion  or  capsule,  as  is  the  case  with  bacteria. 
-The  yeast  cells  are  never  motile. 

Yeast  Protoplasm  and  Cell  Inclusions.— The  contents  of  the 


MORPHOLOGY   AND   RELATIONSHIPS   OF   MICROORGANISMS      39 

yeast  cell  are  more  highly  differentiated  than  are  those  of  bacteria. 
The  ectoplast,  or  limiting  membrane  of  the  protoplasm,  is  easily 
demonstrated  by  plasmolyzing  the  cell.  This  ectoplast  (German 
Hautschicht)  is  the  only  membrane  of  the  young  cell,  and  the 
cell  wall  is  probably  secreted  by  it.  The  protoplasm  is  differ- 
entiated definitely  into  a  nucleus  and  cytoplasm.  The  nucleus 
is  not  as  easily  demonstrated  as  in  the  higher  plants  and  animals,- 


Fig:  19. — Diagram  of  budding  yeast  cells  and  their  contents:  a,  Glycogen 
granules;  b,  vacuoles;  c,  nucleus;  d,  dividing  nucleus  in  bud  formation. 

but  may  be  shown  by  proper  staining  methods.  The  cytoplasm 
usually  contains  one  or  more  vacuoles,  spaces  filled  with  cell  sap 
and  not  taking  the  stain  as  does  the  cytoplasm.  Older  cells 
may  also  show  oil  globules  or  glycogen  granules. 

Reproduction  in  Yeasts. — Yeasts  commonly  multiply  vege- 
tatively  by  budding.  A  bit  of  the  protoplasm  protrudes  on  one 
side  of  the  mother  cell,  the  nucleus  divides,  and  one  part  goes  to 


Fig.  20. — Spores  (ascospores)  of  the  yeast  (Hansen). 

the  bud,  the  other  remains  within  the  cell,  the  bud  enlarges, 
develops  a  cell  wall,  and  is  constricted  off  as  a  distinct  individual. 
Many  yeasts  may  also  under  favorable  conditions  produce  spores. 
The  development  of  the  spores  in  the  yeast  cell  differs  considerably 
from  that  in  the  bacteria.  The  latter  typically  have  but  one  spore 
developed  within  the  cell,  while  a  yeast  cell  usually  produces  two, 
four,  six,  or  even  eight  spores.  The  nucleus  divides  several  times 
to  form  a  number  of  nuclei,  each  of  which,  together  writh  the 


40  VETERINARY   BACTERIOLOGY 

protoplasm  lying  in  contact  with  it,  becomes  surrounded  by  a 
membrane.  A  cell  of  the  yeast  (and  certain  other  fungi)  when 
filled  with  spores  is  called  the  ascus  (pi.  asci)  or  sac.  The  spores 
are  called  ascospores  (Fig.  20).  This  method  of  spore  production 
relates  the  yeasts  quite  definitely  to  some  of  the  higher  fungi. 
In  some  cases  there  is  a  primitive  type  of  sexual  reproduction 
or  fertilization  associated  with  the  development  of  the  spores. 
The  spores  are  more  resistant  to  an  unfavorable  environment  than 
the  vegetative  cells.  When  brought  under  favorable  conditions 
they  germinate  and  develop  into  the  typical  yeast  plant.  In 
old  yeast  cultures  some  cells  develop  heavy  cell  walls,  are  filled 
with  granular  reserve  food  materials,  and  become  potentially 
spores.  Such  cells  are  likewise  probably  resistant  to  unfavorable 
conditions,  and  serve  to  tide  the  yeast  over  periods  of  desiccation 
or  poor  food  supply.  They  resemble  the  chlamydospores  produced 
by  many  molds. 

MORPHOLOGY  OF  THE  HYPHOMYCETES  OR  MOLDS 

The  molds  or  hyphomycetes  do  not  constitute  a  homogeneous 
group  in  the  eyes  of  the  systematic  botanist,  but  belong  to  vari- 
ous subdivisions  of  the  group  of  fungi.  Some  are  related  to 
the  algae  and  are  grouped  under  the  Phy corny cetes,  others  belong 
to  the  sac  fungi  or  Ascomy  cetes,  others  are  related  to  the  smuts, 
rusts,  and  toadstools,  or  Basidiomy cetes,  and  the  largest  number 
belong  to  the  group  of  imperfect  fungi  or  Fungi  Imperfecti.  From 
the  viewpoint  of  the  bacteriologist  these  botanic  relationships 
are  not  significant;  all  the  fungi,  regardless  of  kinship,  that  agree 
in  having  the  plant  body  made  up  of  threads  usually  more  or 
less  branched,  and  forming  more  or  less  loose  or  cottony  masses, 
in  short,  those  that  answer  to  the  popular  conception  of  molds, 
are  grouped  together  as  Hyphomycetes.  Such  a  classification  is 
scientifically  justifiable  only  because  of  the  great  complexity  of 
the  various  members  of  the  family  of  fungi,  and  the  fact  that 
it  is  not  the  systematic  position  but  the  economic  importance  of 
the  forms  that  is  of  significance. 

Form  and  Size  of  Hyphomycetes  or  Molds. — A  mold  may  be 
differentiated  from  the  yeasts  and  bacteria  in  that  it  is  multi- 
cellular,  with  the  cells  united  to  form  more  or  less  bnmrhcd 


MORPHOLOGY   AND   RELATIONSHIPS   OF   MICROORGANISMS      41 

threads  called  hyphce  (sing,  hypha).  The  whole  mass  of  threads 
or  hyphse  which  go  to  make  up  the  plant  of  the  mold  is  called  the 
mycelium.  In  most  molds  certain  threads  are  differentiated 
for  the  production  of  spores.  The  mycelium  of  the  mold  may 
extend  over  a  considerable  area,  growing  deep  into  the  substratum 
for  food  or  into  the  air  to  develop  spores. 

Histology  and  Structure  of  Molds. — The  mycelium  in  some 
molds  is  continuous  throughout  its  length,  possessing  no  cross 
walls  which  might  separate  the  cells  from  each  other.  In  the 
majority  of  forms,  and  in  all  those  of  economic  importance  to 
the  veterinarian,  the  hyphee  are  divided  by  cross  walls  or  septa 


Fig.  21. — Mold  hyphse:  A,  B,  Xon-septate  hyphse  of  the  Phy corny cetes;  C, 
tip  of  a  non-septate  hypha,  showing  numerous  nuclei  and  vacuoles;  D,  septate 
branching  hyphse;  E,  a  single  cell  of  a  septate  hypha,  showing  nucleus  and  vac- 
uoles. 

(sing,  septum).  The  cell  wall  is  composed  of  true  cellulose  in 
a  few  molds,  in  the  majority  it  is  chitinous  as  in  the  bacteria. 
The  almost  universal  presence  of  chitin  in  the  cell  walls  of  the 
fungi  is  frequently  lost  sight  of  by  those  who  regard  its  presence 
in  the  cell  walls  of  bacteria  as  evidence  of  animal  affinities.  The 
protoplasm  of  molds,  as  in  the  yeasts,  is  made  up  of  cytoplasm  and 
nucleus.  The  outer  layer  of  the  cytoplasm  or  ectoplast  is  readily 
demonstrated  in  most  molds  by  plasmolysis.  In  the  forms  that 
do  not  have  septa  dividing  the  hyphse  into  cells,  the  numerous 
nuclei  are  imbedded  in  the  common  cytoplasm.  Functionally 
each  nucleus  with  its  bit  of  surrounding  cytoplasm  constitutes  a 


42  VETERINARY   BACTERIOLOGY 

cell,  although  the  statement  is  often  made  that  the  entire  mold 
filament  in  the  non-septate  type  is  a  single  cell. 

Reproduction  of  Molds. — It  is  impracticable  to  go  into  detail 
concerning  the  reproduction  of  molds.  Spores  of  many  different 
types  are  produced  (Fig.  22),  sometimes  three  or  four  kinds  by 
a  single  species.  The  spores  exhibit  every  conceivable  shape  and 
coloring,  are  sometimes  unicellular,  at  other  times  multiseptate. 
Hundreds  of  genera  and  thousands  of  species  are  known.  The 
names  applied  to  the  different  parts  of  the  molds  concerned  in 
reproduction  and  the  manner  in  which  the  spores  are  borne  in 
some  of  the  commoner  molds  may,  however,  be  briefly  discussed. 

Molds  may  be  divided,  for  convenience,  into  those  which 
bear  the  spores  enclosed  in  a  spore  case  or  sporangium  and  those 
in  which  they  are  not  so  inclosed.  This  sporangium  is  commonly 


Fig.  22. — Types  of  mold  spores. 

borne  at  the  tip  of  a  hyphal  thread  differentiated  for  the  pur- 
pose, called  a  sporangiophore.  Spores  not  produced  inside  of 
a  sporangium  and  not  the  result  of  fertilization  (i.  e.,  asexual 
spores)  are  termed  conidia  (sing,  conidiwri).  Conidia  are  usually 
developed  at  the  tip  of  specialized  branches  called  conidiophores. 
Sometimes  they  are  formed  by  the  breaking  up  of  the  mycelial 
threads  or  hyphae,  and  are  then  called  o'idia  (sing,  o'idium), 
in  other  cases  they  develop  within  the  hyphae  and  are  surrounded 
by  it  as  by  a  sheath.  When  one  of  the  cells  in  a  hypha  becomes 
enlarged  and  surrounded  with  a  heavy  wall  it  is  called  a  chlamydo- 
spare.  Some  molds  develop  spores  as  a  result  of  the  union  of 
sex  cells  (sexual  spores).  These  are  called  ascospores  when  pro- 
duced in  sacs  (asci)  and  zygospores  when  formed  by  the  union  of 
two  like  cells  as  in  certain  Phycomycetes. 

Spores  of  the  molds  are  commonly  born  on  hyphae  that  extend 


MORPHOLOGY   AND   RELATIONSHIPS   OF   MICROORGANISMS      43 

into  the  air  away  from  the  moist  surface  of  the  medium  in  which 
they  are  growing.     This  facilitates  their   dispersal  by  the   air 


Fig.  23. — Types  of  the  spores  and  the  spore-bearing  organs  of  the  molds — 
1,  Sporangium  of  the  Mucor:  a,  columella;  6,  sporangiophore;  c,  spores;  d, 
sporangium  wall.  2,  Sporangia  of  Sporodinia:  a,  sporangiophore;  6,  sporangia 
containing  spores.  3.  Ascus  of  an  ascomycete,  Peziza:  a,  ascus  or  spore  sac; 
6,  spore;  c,  sterile  threads  or  paraphyses.  4,  Oi'dium  spore  formation;  thet 
hyphse  are  segmenting  to  form  spores  or  oidia.  5,  a,  Chlamydospores  formed 
in  the  hypha  of  a  Chlamydomucor.  6,  Zygospore  of  a  Mucor:  a,  hypha;  6, 
suspensor;  c,  zygospore.  7,  Conidiophore  and  conidia  of  Penicillium:  a,  con- 
idiophore;  6,  verticillate  branches  of  the  conidiophore;  c,  chains  of  spores  or 
conidia.  8,  Aspergillus:  a,  conidiophore;  6,  inflated  tip  of  the  conidiophore;  c, 
sterigmata;  d,  chain  of  spores.  9,  a,  Hypha;  6,  poorly  differentiated  conidio- 
phore; c,  chain  of  conidia. 

currents.     When  they  fall  upon  a  suitable  medium  they  ger- 
minate and  soon  develop  the  typical  mold  plant. 

MORPHOLOGY  OF  THE  PROTOZOA 

The  protozoa  are  unicellular  and  bear  much  the  same  relation 
to  the  higher  animals  or  metazoa  that  the  bacteria  do  to  the  higher 
plants.  Notwithstanding  that  they  are  reckoned  among  the 
simplest  forms  of  life,  they  are,  nevertheless,  greatly  diversified 
in  shape,  size,  and  structure.  Only  the  barest  outline  of  their 
structure  can  be  given  here.  For  a  more  detailed  account  the 
student  is  referred  to  the  section  on  Protozoa. 


44  VETERINARY  BACTERIOLOGY 

Form  and  Size  of  Protozoa. — The  pathogenic  protozoa  vary 
in  size  from  those  visible  to  the  naked  eye  to  those  barely  visible 
with  the  highest  powers  of  the  microscope.  Some  are  undoubt- 
edly ultramicroscopic,  the  organism  causing  yellow  fever,  for 
example.  In  form  and  shape  the  greatest  diversity  is  to  be  noted. 
Some  are  without  definite  shape,  and  are  apparently  only  lumps  of 
protoplasm,  others  are  highly  differentiated  and  have  as  great 
variety  of  organs  (organella)  as  some  of  the  higher  animals. 

Histology. — A  true  cell  wall,  as  found  in  the  bacteria,  yeasts, 
and  fungi,  is  frequently  not  present  in  the  protozoa.  When 
found,  it  is  chitinous  in  nature.  The  ectoplast  forms  the  limit- 
ing membrane  of  the  cell  in  the  majority  of  cases.  The  protoplasm 
is  differentiated  into  nucleus  (sometimes  two,  a  micronudeus 
and  a  macronucleus)  and  endoplasm  or  cytoplasm.  Power  of  move- 
ment is  possessed  by  many  forms.  This  may  be  due  to  the 
development  of  pseudopodia,  of  flagella,  or  of  cilia. 

Reproduction. — Asexual  reproduction  is  accomplished  in  many 
cases  by  a  simple  process  of  fission,  in  others  the  procedure  is 
much  more  complex.  Sexual  reproduction  is  quite  general,  but 
here  again  the  complexity  is  so  great  as  to  render  brief  treatment 
impracticable.  The  relationship  and  structure  of  these  forms 
will  be  considered  in  greater  detail  under  the  heading  of  Patho- 
genic Protozoa  in  Section  V. 


CHAPTER  III 

PHYSIOLOGY  OF  MICROORGANISMS 

PHYSIOLOGY  has  been  defined  by  Barnes  to  include  "  a  study 
of  the  behavior  of  plants  (and  animals)  of  all  sorts,  and  of  the 
ways  in  which  this  is  affected  by  external  agents  of  every  sort." 
In  our  discussion  of  the  physiology  of  microorganisms  we  shall 
have  to  deal  principally  with  the  interrelationships  existing 
between  these  microorganisms  and  their  environment. 

FOOD  RELATIONSHIPS  OF  MICROORGANISMS 
A  food  is  any  substance  which  a  living  organism  may  make 
a  part  of  its  living  material  or  use  as  a  source  of  growth  energy. 
The  term  is  frequently  used  very  loosely  to  include  all  the  sub- 
stances of  which  an  organism  may  make  any  use.  For  example,  a 
distinction  is  sometimes  made  between  green  plants  and  animals 
on  the  basis  of  food  used.  The  former  are  said  to  live  on  inorganic 
foods  and  the  latter  on  organic.  This  distinction  is  erroneous. 
The  difference  is  simply  that  green  plants  can  manufacture  their 
own  foods  out  of  inorganic  material  by  the  aid  of  the  energy 
secured  from  the  sun's  rays  through  the  green  coloring-matter  or 
chlorophyll,  while  animals  make  use  of  food  already  prepared. 
The  materials  of  which  some  microorganisms  make  use  are  no  more 
foods  than  the  rays  of  the  sun  are  a  food  for  green  plants. 

Composition  of  the  Cell. — The  food  utilized  by  any  micro- 
organism must  contain  the  elements  needed  for  the  building  up 
of  the  cell  substance.  The  analysis  of  such  cells  shows  them 
to  be  made  up  of  the  same  elements  as  those  of  higher  plants 
and  animals,  namely,  carbon,  oxygen,  nitrogen,  hydrogen,  and 
smaller  amounts  of  phosphorus,  iron,  magnesium,  calcium,  and 
some  other  elements.  The  foods  utilized  by  organisms  must, 
therefore,  contain  these  elements  likewise. 

Sources  and  Kinds  of  Foods. — Some  bacteria,  like  the  green 
plants,  are  capable  of  manufacturing  their  own  food.  For  this 
purpose  a  source  of  energy  is  necessary.  Some  species  utilize  the 

45 


4G  VETERINARY   BACTERIOLOGY 

energy  of  the  rays  of  sunlight  in  much  the  same  manner  probably 
as  do  green  plants.  The  coloring-matter  in  these  forms,  however, 
is  purple  or  red  (bacteriopurpuriri) .  Other  forms  living  in  water 
which  contains  hydrogen  sulphid,  as  in  the  sulphur  springs,  oxidize 
the  hydrogen  sulfid  to  free  sulphur  and  even  sulphuric  acid  and 
gain  energy  for  the  manufacture  of  their  foods  from  carbon  dioxid, 
water,  and  other  compounds  by  this  process.  Still  other  forms 
are  believed  to  make  use  of  iron,  ammonia,  nitrites,  and  other 
inorganic  substances,  and  by  their  oxidation  secure  the  necessary 
energy.  Organisms  which  can  manufacture  their  own  food  out 
of  inorganic  substances  are  said  to  be  prototrophic.  The  proto- 
trophic  microorganisms  so  far  as  known  are  all  bacteria  or  molds. 
Most  microorganisms  utilize  organic  matter  in  a  dead  or  living 
condition  for  food.  Those  which  utilize  dead  organic  matter  are 
called  metatrophic,  while  those  requiring  living  material  or  complex 
protein  foods  are  called  paratrophic.  The  latter  are  frequently  dis- 
ease producing.  It  must  not  be  supposed  that  these  division  lines 
are  strictly  drawn.  For  example,  certain  bacteria  seem  to  make  use 
of  the  oxidation  of  carbohydrates  and  other  organic  substances  to 
enable  them  to  take  up  the  nitrogen  from  the  air  and  to  convert  it 
into  usable  form.  Such  are  both  prototrophic  and  metatrophic. 

The  peculiar  food  requirements  of  the  different  species  must 
be  kept  in  mind  in  the  preparation  of  nutrient  media  for  their 
growth.  Some  organisms  will  not  grow  in  the  presence  of  organic 
materials,  while  others  require  such  specialized  media  as  blood- 
serum  or  hemoglobin. 

A  second  grouping  of  microorganisms  commonly  used  is  based 
upon  the  relationship  to  other  living  organisms.  Those  which 
do  not  require  a  living  host  (animal  or  plant)  are  called  sapro- 
phytes if  bacteria,  yeasts,  or  molds,  and  saprozoites  if  protozoa; 
those  which  require  a  living  host  are  called  parasites.  Those 
parasites  which  do  not  produce  disease  are  termed  commensals. 

MOISTURE  RELATIONSHIPS  OF  MICROORGANISMS 

Microorganisms  require  considerable  amounts  of  water  for 
their  development.  The  optimum  condition  for  growth  in 
most  cases  is  saturation.  There  is  great  variation  in  ability 
to  resist  desiccation  (drying).  The  spores  of  some  bacteria  and 


PHYSIOLOGY   OF   MICROORGANISMS  47 

fungi  and  the  encysted  cells  of  some  protozoa  will  live  for  years, 
while  other  forms  are  destroyed  if  allowed  to  become  completely 

dried. 

RESPIRATION  OF  MICROORGANISMS 

Respiration  is  frequently  defined  as  the  taking  up  of  oxygen 
and  the  elimination  of  carbon  dioxid.  This  definition  is  entirely 
inadequate  when  we  come  to  a  discussion  of  microorganisms, 
if,  indeed,  it  can  be  applied  in  any  case  even  to  higher  animals 
and  plants'.  Respiration  seems  fundamentally  to  be  the  process 
whereby  energy  is  generated  in  the  cell.  Energy  when  evolved  in 
the  cell  always  originates  from  chemical  changes  in  the  compounds 
within  the  cell.  Whether  or  not  this  energy  may  be  gained  by 
the  oxidation  of  food  materials  when  taken  into  the  cell,  or  whether 
they  must  be  first  built  up  into  protoplasm  and  this  then  broken 
down,  is  a  matter  of  dispute  at  present  among  scientists.  In 
any  event  the  presence  of  free  oxygen  is  certainly  not  neces- 
sary to  this  release  of  energy,  for  many  bacteria  as  well  as  other 
plants  and  animals  live  in  the  absence  of  free  oxygen.  Organ- 
isms that  grow  only  in  the  presence  of  oxygen  are  called  aerobic; 
those  which  will  grow  only  in  the  absence  of  free  oxygen,  anaerobic, 
and  those  which  will  grow  either  with  or  without  free  oxygen,  facul- 
tative. It  is  probable  that  most  of  the  so-called  anaerobes  grow 
better  in  the  presence  of  minute  quantities  of  oxygen.  The 
end-products  of  respiration  are  found  to  differ  with  the  type, 
aerobic  bacteria  usually  produce  carbon  dioxid  and  waterj  anae- 
robic forms,  less  highly  oxidized  substances,  such  as  alcohol  and 
butyric  acid. 

The  oxygen  requirements  of  anaerobic  bacteria  must  be  recog- 
nized in  the  laboratory  if  they  are  to  be  successfully  cultivated. 
The  air  of  the  culture-tube  or  flask^jnay  be  removed  by  a  stream 
of  hydrogen,  nitrogen,  or  some  other  inert  gas,  the  oxygen  may  be 
absorbed  by  the  use  of  alkaline  sodium  pyrogallate,  the  air  may 
be  exhausted  by  an  air-pump,  the  oxygen  may  be  excluded  by 
covering  the  medium  with  oil  or  some  similar  material,  or  the 
organism  may  be  mixed  with  some  aerobic  form  which  will  use 
up  the  oxygen  and  allow  growth  of  the  anaerobe.  Probably 
the  latter  is  the  common  method  whereby  anaerobes  are  able  to 
grow  in  nature. 


48  VETERINARY  BACTERIOLOGY 

TEMPERATURE  RELATIONSHIPS  OF  MICROORGANISMS 

Optimum  Temperature. — The  optimum  growth  tempera- 
ture is  that  which  most  favors  the  development  of  the  micro- 
organism. The  optimum  varies  with  the  species.  A  few  organisms 
found  in  the  ocean,  in  cold  waters,  alpine  regions,  etc.,  prefer  a 
low  temperature,  from  0°  to.  15°.  These  are  called  psychrophilic. 
Those  which  prefer  a  somewhat  higher  temperature  are  called 
mesophilic.  These  latter  may  be  again  subdivided  into  those  that 
prefer  a  "  room  "  temperature  of  18°  to  25°,  and  those  that 
prefer  blood  heat  (man  37.5°)  for  the  most  parasitic  forms.  Tem- 
peratures such  as  are  found  in  hot  springs,  interior  of  compost 
heaps  (50°  to  70°)  favor  the  development  of  thermophilic  bacteria. 

Minimum  Temperature. — The  lowest  temperature  at  which 
an  organism  will  continue  growth  is  said  to  be  its  minimum. 
This  temperature  varies  for  different  species.  Some  organisms 
will  multiply  in  brine  held  at  temperatures  lower  than  the  freez- 
ing-point of  water. 

Maximum  Growth  Temperature. — The  highest  temperature 
at  which  an  organism  will  multiply  is  called  its  maximum.  This 
must  not  be  confused  with  the  thermal  death  point  (see  below). 
The  majority  of  bacteria  cannot  grow  above  45°. 

Growth  Temperature  Range. — The  differences  between  the 
minimum  and  maximum  growth  temperatures  vary  within  rather 
wide  limits.  Those  organisms  which  exhibit  considerable  adap- 
tability and  are  able  to  grow  through  a  wide  range  of  temperature 
are  called  eurythermic.  Most  of  the  saprophytic  organisms  belong 
here.  The  parasitic  types  which  have  minima  and  maxima 
varying  but  little  from  the  optima  are  stenothermic. 

Thermal  Death  Point. — The  thermal  death  point  of  an  organism 
is  that  temperature  which  under  given  conditions  will  certainly 
destroy  all  the  cells.  The  following  factors  must  be  taken  into 
consideration  in  the  determination  of  any  thermal  death  point: 

1.  The  Absence  or  Presence  of  Spores. — Spores  are  much  more 
resistant  to  high  temperatures  than  the  vegetative  cells.     Forms 
having  spores,  therefore,  have  two  thermal  doath  points,  one  for 
the  vegetative  cells  and  the  other  for  the  spon  •>. 

2.  Presence  or  Absence  of  Moisture. — Bacteria  are  more  resistant 
to  dry  than  to  moist  heat.     The  thermal  death  point  is  probably 


PHYSIOLOGY   OF   MICROORGANISMS  49 

the  temperature  at  which  incipient  coagulation  of  the  albuminous 
protoplasm  occurs,  resulting  in  an  inability  to  function.  Water 
is  necessary  for  this  coagulation.  The  following  table  from  Frost 
and  McCampbell  illustrates  this  point: 

Egg  albumen  +  50  per  cent,  water  coagulates  at  56°  C. 
Egg  albumen  +  25         "  74-80°  C. 

Egg  albumen  +  18         "  "  80-90°  C. 

Egg  albumen  +     6  "  145°  C. 

Egg  albumen  +  no  water  "        160-170°  C. 

This  fact  is  emphasized  by  the  laboratory  methods  of  sterilization. 
The  autoclave,  with  live  steam  at  temperature  of  120°,  will  destroy 
in  ten  minutes  the  most  resistant  spores,  while  in  the  hot-air 
oven  a  temperature  of  150°  to  170°  for  an  hour  is  necessary. 

3.  Reaction  and  Composition  of  Medium. — The  reaction  and 
composition  of  the  medium  has  been  found  to  exert  a  marked  influ- 
ence on  the  thermal  death  point.     In  comparative  work,  care 
must  be  exercised  to  use  media  of  uniform  reaction  and  com- 
position. 

4.  Time  of  Exposure. — In  general,  the  higher  the  temperature 
the  shorter  the  period  required  to  destroy  life  in  the  cells.     Math- 
ematic  formulas  have  been  developed  giving  the  time  as  a  func- 
tion of  temperature  for  some  forms. 

5.  Specific    Character    of   Organism. — Intrinsic    variations    in 
the  character  of  protoplasm  of  different  species  make  it  necessary 
to  determine  the  thermal  death  point  for  each  species. 

LIGHT  RELATIONSHIPS  OF  MICROORGANISMS 

A  few  bacteria  possessing  bacteriopurpurin  require  light 
for  their  development.  For  most  other  microorganisms,  particu- 
larly the  bacteria  and  the  pathogenic  protozoa,  darkness  is  the 
optimum  light  condition.  Light,  particularly  the  direct  rays  of  the 
sun,  will  destroy  all  but  the  most  resistant  bacteria  if  .exposed 
for  a  sufficient  length  of  time.  Sunlight  when  passed  through 
a  prism  is  readily  broken  up  into  its  constituent  colors,  the  least 
refracted  rays,  or  the  reds  and  yellows,  at  one  end,  and  the  more 
highly  refracted  rays,  the  blues  and  the  violets,  at  the  other. 
Exposure  of  bacteria  to  these  various  colored  rays  has  shown 

4 


50  VETERINARY    BACTERIOLOGY 

the  blues  and  violets  to  be  most  powerful,  while  the  reds  and 
yellows  of  the  other  end  have  little  or  no  effect.  It  will  be  re- 
membered that  the  blue  and  violet  rays  are  the  ones  which  affect 
the  photographic  plate  most  intensely.  The  germicidal  action 
of  light  on  the  pathogenic  bacteria  is  of  the  greatest  practical 
importance.  It  renders  infection  through  the  medium  of  the  air 
in  most  cases  a  remote  possibility. 

Effect  of  Electricity  on  Bacteria 

Strong  direct  currents  of  electricity  passed  through  a  solution 
containing  bacteria  will  sterilize  it.  It  is  difficult,  however, 
to  dissociate  the  physical  effect  of  the  current  directly  upon  the 
bacteria  from  the  action  of  the  chemicals  produced  by  electrolysis. 
No  practical  use  has  been  made  of  the  destructive  action  of 
electricity  upon  microorganisms,  as  the  method  is  difficult  to  apply 
and  is  inefficient  at  best.  The  Rontgen  rays  (x-rays)  do  not 
destroy  bacteria  even  when  the  latter  are  exposed  for  consid- 
erable periods. 

RELATIONSHIPS  OF  MICROORGANISMS. TO  CHEMICALS 

Microorganisms  are  profoundly  affected  both  in  growth  and 
movement  by  the  chemicals  with  which  they  come  in  contact. 
They  may  be  attracted  or  repulsed,  stimulated  to  increased  growth, 
their  development  inhibited, or  they  may  be  destroyed  when  certain 
substances  are  present. 

Chemotaxy. — Motile  microorganisms  are  attracted  or  repulsed 
by  certain  chemicals.  The  first  is  known  as  positive  chemotaxy, 
the  latter  as  negative.  Certain  protozoa  and  bacteria  are  attracted 
by  oxygen  and  may  be  observed  to  swim  about  air  bubbles  under 
the  microscope.  From  their  movements  it  is  evident  that  differ- 
ent species  prefer  varying  amounts  of  oxygen.  This  results  in  a 
grouping  of  the  different  kinds  in  concentric  circles  about  the 
bubble.  This  type  of  chemotaxy  is  called  curotaxy  (not  ae'ro- 
tropism) .  The  avidity  of  certain  bacteria  for  oxygen  has  been  used 
in  the  laboratory  for  their  isolation  from  water,  particularly  the 
Asiatic  cholera  organisms.  Peptones  and  meat  extractives  at- 
tract many  kinds  of  bacteria.  This  phenomenon  may  be  rcndily 
demonstrated  by  introducing  the  tip  of  a  minute  capillary  tube 


PHYSIOLOGY    OF   MICROORGANISMS 


51 


filled  with  such  a  solution  into  water  containing  numerous  bacteria. 
These  will  be  found  to  congregate  in  great  numbers  about  the 
mouth  of  the  tube  and  to  enter  it.  Probably  chemotaxis  accounts 
for  the  flocking  of  the  leukocytes  or  white  blood  corpuscles  to 


Fig.  24. — Chemotaxy:  a,  Spirilla  attracted  by  a  green  algal  cell  which  is 
giving  off  oxygen,  aerotaxis;  6,  a  leukocyte  containing  several  bacteria  which 
it  has  engulfed;  c,  capillary  pipette  containing  a  solution  of  beef  extract,  and 
at  2  an  air  bubble,  placed  in  a  drop  of  water  containing  motile  bacteria. 
The  latter  are  attracted  in  large  numbers  to  the  mouth  of  the  tube;  d,  an  air 
bubble  surrounded  by  two  concentric  circles  of  organisms,  the  inner  one 
bacteria,  the  outer  protozoa.  Each  remains  in  the  concentration  of  oxygen 
most  favorable  to  its  growth. 

any  part  of  the  body  attacked  by  certain  bacteria.  Micro- 
organisms are  not  always  attracted  by  food  stuffs  and  repelled 
by  harmful  substances.  A  mixture  of  peptone  and  mercuric 
chlorid  will  attract  bacteria  and  then  destroy  them. 


Fig.  25. — Chemotropism:  a,  6,  Mold  hyphse  and  conidiophores,  showing  the 
negative  hydrotropism  of  the  latter;  c,  an  air  bubble  in  a  medium  with  four 
germinating  mold  spores.  The  hyphae  are  growing  toward  the  air,  showing 
positive  aerotropism. 

Tropisms. — Organisms  which  are  not  free  to  move  in  response 
to  a  chemotactic  stimulus  may  nevertheless  be  influenced  in  their 
direction  of  growth.  Such  a  response  in  the  direction  of  growth 


52  VETERINARY   BACTERIOLOGY 

to  an  external  stimulus  is  called  a  tropism.  Mold  hyphae  will 
often  grow  toward  a  moist  medium,  while  the  conidiophores  which 
they  bear  rise  at  right  angles  to  its  surface  and  seek  to  produce 
the  spores  as  far  as  possible  from  a  moist  surface.  These  phenom- 
ena are  known  respectively  as  positive  and  negative  hydrotropism. 
The  influencing  of  the  direction  of  the  growth  by  the  action  of 
chemicals  is  called  chemotropism.  The  forms  of  mold  and 
bacterial  colonies,  when  growing  upon  artificial  media,  are 
largely  determined  by  this  factor.  For  example,  many  molds 
radiate  in  practically  straight  lines  from  their  point  of  origin  in 
a  medium, and  every  branch  quite  exactly  bisects  the  angle  between 
the  two  filaments  on  either  side.  This  mutual  repulsion  of  the 
hyphse  is  doubtless  due  to  certain  of  the  products  excreted  by  the 
cells.  Heliotropism,  or  the  influence  of  light  on  the  direction  of 
growth,  is  also  observed  in  some  forms.  J 

Influence  of  Reaction  of  Medium  on  Growth. — Many  organisms 
are  quite  exacting  in  their  requirements  as  to  the  reaction  of  the 
medium  in  which  they  are  grown.  Some  forms  grow  best  in  a 
medium  slightly  acid,  others  refuse  to  develop  except  in  one  which 
is  slightly  alkaline.  The  majority  of  bacteria,  however,  grow  well 
in  a  medium  that  is  neutral  to  litmus. 

ANTISEPTICS  AND  DISINFECTANTS 

An  antiseptic  is  anything  that  will  inhibit  the  growth  of  micro- 
organisms without  necessarily  destroying  them.  In  the  broadest 
sense,  this  term  would  include  such  physical  agencies  as  the  action 
of  cold  and  heat,  but  in  practice  it  is  generally  confined  to  chemical 
substances.  A  disinfectant  is  a  substance  that  will  destroy  patho- 
genic bacteria.  Inasmuch  as  pathogenic  and  non-pathogenic 
bacteria  are  both  destroyed  by  the  same  substances,  there  is 
little  real  difference  in  the  meaning  of  disinfectant  and  germicide 
(something  that  will  destroy  all  bacteria).  A  deodorant  is  any 
substance  that  masks  disagreeable  odors  or  eliminates  them  entirely 
by  removing  their  cause.  A  deodorant  may  or  may  not  be  a  dis- 
infectant or  antiseptic.  These  latter  terms  are  relative  ones  only, 
for  any  disinfectant  if  sufficiently  diluted  becomes  an  antiseptic. 

Theories  of  Action  of  Antiseptics  and  Disinfectants. — Germ- 
icides may  destroy  microdrganisma  by  forming  compounds  with 


PHYSIOLOGY   OF   MICROORGANISMS  53 

the  protoplasm,  by  dissolving  or  coagulating  the  protoplasm,  or  by 
oxidation  and  complete  destruction  of  the  cells.  Our  most  efficient 
disinfectants  are  those  which  destroy  in  the  manner  first  named. 
The  activity  and  efficiency  of  many  disinfectants  depend  upon 
the  ionization  of  the  compound  in  solution.  This  is  particularly 
true  with  the  salts  of  heavy  metals,  such  as  mercuric  chlorid 
(corrosive  sublimate).  It  is  the  ionized  mercury  that  is  poison- 
ous to  bacteria.  Mercuric  chlorid  does  not  ionize  in  pure  alcohol, 
hence  it  is  not  poisonous  to  bacteria  when  in  solution  in  that 
substance.  The  addition  of  various  other  chemicals  may  increase 
or  decrease  the  ionization  of  the  disinfectant  and  enhance  or  dimin- 
ish its  destructive  action. 

Disinfectants  and  Antiseptics  in  Common  Use. — Salts  of  the 
Heavy  Metals. — The  salts  of  gold,  silver,  copper,  and  mercury 
are  all  active  disinfectants.  Copper  sulphate  is  sometimes 
used  in  an  effort  to  free  water  reservoirs  and  other  city  supplies 
from  growths  of  objectionable  algae.  Mercury  is  the  most  efficient 
of  the  metals  and  its  salts  are  most  commonly  used.  It  acts  by 
forming  a  mercuric,  albuminate  of  the  protoplasm.  When  used 
in  any  solution  containing  considerable  quantities  of  protein  or 
similar  materials,  as  in  feces,  it  must  be  used  in  excess  and  thor- 
oughly mixed,  for  it  is  apt  to  form  an  insoluble  coating  over  the 
surface  of  the  solid  particles  and  protect  the  bacteria  in  the  interior 
from  destruction.  Mercuric  chlorid  is  usually  used  in  solutions 
of  1  :  1000  or  1  :  2000. 

Lime,  unslaked,  is  a  fairly  efficient  disinfectant.  It  is  par- 
ticularly useful  because  in  the  form  of  whitewash  it  may  be 
applied  as  a  permanent  coating  to  the  walls  of  stables  and  out- 
buildings. Feces  and  urine  may  be  disinfected  by  a  mixture 
of  equal  parts  of  a  20  per  cent,  solution  of  freshly  slaked  lime 
with  the  material  to  be  disinfected. 

Phenol,  or  carbolic  acid,  C6H5OH,  and  the  methyl  phenols  or 
cresols,  CsFLCHaOH,  either  pure  or  in  the  trade  mixtures,  such 
as  kreso,  tricresol,  creolin,  etc.,  are  among  the  most  efficient 
and  useful  of  disinfectants.  They  are  most  frequently  used  in 
1  to  5  per  cent,  solutions,  and  will  destroy  bacteria  even  in  the 
presence  of  quantities  of  organic  matter. 

Sulphur    Dioxid    and    Sulphurous    Add. — When    sulphur    is 


54  VETERINARY   BACTERIOLOGY 

burned  it  yields  sulphur  dioxid,  a  gas  that  has  been  much  used 
in  fumigation.  It  is  powerless  to  destroy  bacteria  unless  moisture 
is  present,  with  which  it  may  unite  and  form  sulphurous  acid. 
The  latter  is  a  very  active  bleaching  and  corrosive  agent,  hence 
it  should  not  be  used  except  where  it  can  do  no  harm.  One 
pound  of  water  (about  1  pint)  should  be  vaporized  in  a  room 
for  every  5  pounds  of  sulphur  burned.  This  amount  should 
efficiently  disinfect  1000  cubic  feet.  Insects  and  other  vermin 
are  destroyed  by  the  sulphur  fumes. 

Formaldehyd,  HCHO. — Formaldehyd  is  the  gas  used  most 
widely  in  fumigation  and  disinfection.  It  is  very  soluble  in 
water  and  is  commonly  sold  as  formalin,  a  40  per  cent,  solution 
of  formaldehyd.  Like  sulphur  dioxid,  formaldehyd  is  efficacious 
only  in  the  presence  of  moisture,  but,  unlike  it,  does  not  bleach 
fabrics  or  injure  materials.  Formaldehyd  may  be  evolved  in 
gaseous  form  for  disinfection  in  a  variety  of  ways.  Incomplete 
combustion  of  methyl  alcohol  according  to  the  reaction 

2CH3OH  +  02  =  2HCHO  +  2H2O 

is  utilized  in  a  number  of  lamps  upon  the  market.  When  properly 
carried  out  the  method  may  be  efficient,  but  it  has  several 
disadvantages,  i.  e.,  expense  and  presence  of  a  fire  in  a  closed 
room.  Heating  the  formalin  over  an  open  flame  will  liberate  a 
part  of  the  formaldehyd  readily,  but  under  these  conditions 
it  polymerizes  and  some  of  the  polymers  (paraformaldehyd)  are 
insoluble.  If  the  evaporation  is  continued  to  dryness,  all  of  these 
will  again  be  broken  up  and  given  off  as  formaldehyd.  The 
same  result  can  be  reached  more  quickly  by  the  addition  of  glyc- 
erin or  some  salt  which  will  raise  the  boiling-point  of  the  solu- 
tion above  the  dissociation  temperature  of  the  paraformaldehyd. 
An  autoclave  or  closed  vessel  in  which  the  solution  is  heated  con- 
siderably above  the  boiling-point  of  water  will  serve  the  same  pur- 
pose. Twelve  ounces  of  formalin  should  be  used  for  every  1000 
cubic  feet  to  !><>  fumigated.  A  convenient  method  for  fumigating 
small  rooms  is  to  pour  formalin  over  crystals  of  potassium  per- 
manganate in  an  earthen  vessel  that  is  a  poor  conductor  of  heat. 
The  permanganate  is  an  active  oxidizing  agent  and  converts  part 
of  the  formaldehyd  into  carbon  dioxid  and  water,  with  the 


PHYSIOLOGY   OF   MICROORGANISMS  55 

liberation  of  sufficient  heat  to  vaporize  a  large  portion  of  >  the 
remainder.  The  solid  paraformaldehyd  or  paraform  may  be 
heated  and  is  thereby  converted  into  formaldehyd  gas.  On 
account  of  its  cheapness  and  effectiveness  formaldehyd  is  used 
much  more  commonly  at  present  than  any  other  of  the  gaseous 
disinfectants. 

Adjustment  of  Organisms  to  Osmotic  Pressure. — Any  crystal- 
loid in  solution  behaves  within  the  limits  of  the  solution  like  a 
gas,  and  the  same  laws  of  diffusion  and  diffusion  pressures  are 
applicable.  Every  organism  when  growing  is  surrounded  by 
water  containing  substances  in  solution,  and  it  also  contains 
certain  salts  dissolved  in  the  "  cell  sap  "  or  water  in  the  pro- 
toplasm. The  ectoplast  or  limiting  membrane  of  the  protoplasm 
lying  just  within  the  cell  wall  is  certainly  in  most,  probably  all, 
cases  a  semipermeable  membrane,  i.  e.,  it  will  allow  some  sub- 
stances to  pass  through  readily,  as  water;  others  pass  through 
slowly,  and  still  others,  although  in  true  solution,  cannot  pass  at 
all.  This  ectoplast,  in  short,  serves  as  an  osmotic  membrane 
and  determines  what  substances  may  enter  and  leave  the  cell. 
An  active  cell  always  maintains  within  its  sap  a  greater  con- 
centration of  solutes  than  the  surrounding  medium,  the  pressure 
on  the  inside  of  the  membrane  is  greater  than  on  the  outside, 
and  the  cell  is  said  to  be  in  a  state  of  turgor.  When  such  a  cell 
is  placed  in  water  containing  a  greater  percentage  of  solutes  than 
does  the  cell  sap,  water  leaves  the  latter  until  the  concentra- 
tion on  the  inside  and  outside  again  becomes  the  same.  This 
means  a  shrinking  of  the  protoplasm;  it  withdraws  from  the  cell 
wall  and  the  cell  is  said  to  be  plasmolyzed.  After  a  time  the 
cell  may  readjust  the  amount  of  solutes  in  the  cell  sap  and  regain 
its  turgor.  For  every  cell,  however,  there  is  a  limit  beyond 
which  the  organism  cannot  go.  Some  yeast  cells  have  been  found 
to  develop  slowly  in  a  solution  cbntaining  35  per  cent,  of  cane- 
sugar.  A  solution  of  this  concentration  exerts  a  pressure  of  more 
than  350  pounds  per  square  inch.  It  is  apparent  that  such  a  cell 
must  be  profoundly  modified.  The  fact  that  concentration 
of  solutes  inhibits  the  growth  of  microorganisms  is  utilized  in 
the  preservation  of  many  food  stuffs.  Such  foods  as  syrups, 
jellies,  and  candied  fruits  are  preserved  by  the  high  osmotic 


56  VETERINARY   BACTERIOLOGY 

pressure  of  the  solutes.  The  action  of  sugars,  salts,  etc.,  is  in 
the  nature  of  a  physical  antiseptic.  Physiological  salt  solution 
is  one  having  the  same  concentration  of  salt  as  do  the  body  cells 
of  the  particular  organism  to  be  studied.  It  usually  contains 
.85  per  cent,  of  sodium  chlorid. 

SYMBIOSIS,  ANTIBIOSIS,  AND  COMMENSALISM 

Two  organisms  that  live  together  and  which  are  mutually 
beneficial  are  said  to  live  in  symbiosis.  Each  organism  is  called 
a  symbion  or  symbiont.  The  symbionts  are  not  necessarily  closely 
related  forms  and  may  belong  to  the  most  widely  separated  groups 
of  plants,  as,  for  example,  bacteria  and  members  of  the  bean  family 
of  the  flowering  plants. 

Antibiosis  is  that  condition  which  obtains  when  organisms 
prove  inimical  to  each  other's  development.  The  growth  of  one 
species  of  organism  in  a  culture-medium  may  completely  inhibit 
the  development  of  some  other  type.  For  example,  the  organism 
(Streptococcus  lacticus)  which  ordinarily  sours  milk  prevents  the 
development  of  most  other  species. 

An  organism  which  uses  the  by-products  of  another  as  food, 
in  other  words,  is  parasitic  without  producing  disease,  is  called  a 
commensal.  Many  of  the  bacteria  found  on  the  skin,  in  the  mouth, 
and  in  the  intestinal  tract  of  man  and  animals  are  of  this  character. 

All  degrees  of  intergradation  between  symbiosis,  true  para- 
sitism, and  commensalism  have  been  described  for  different 
species. 

PIGMENT  PRODUCTION  BY  MICROORGANISMS 

Molds,  yeasts,  and  bacteria  are  frequently  found  to  be  chromo- 
genic,  that  is,  capable  of  producing  pigments  or  coloring-matter. 
The  colors  produced  range  through  all  the  colors  and  even  shades 
of  color  of  the  spectrum.  A  few  only  of  the  pathogenic  forms 
produce  pigments.  Many  organisms,  particularly  molds  and 
bacteria,  excrete  a  pigment-forming  substance  which  diffuses 
through  the  culture-medium  and  colors  it.  Such  an  organism 
is  the  Bacillus  pyor>/<in(  //*,  which  produces  a  diffuse  pigment, 
one  that  changes  the  medium  first  to  a  green,  then  to  a  brown. 
Some  organisms  produce  pigment  granules  outside  the  cell,  such 
are  said  to  be  chromoparous,  for  example,  Bacillus  prodigiosus, 


PHYSIOLOGY   OF   MICROORGANISMS 


57 


which  produces  a  red  pigment.     The  cell  walls  of  some  bacteria 
(such  as  B.  violaceus)  and  of  many  of  the  molds  are  colored. 

Pigments  are  generally  produced  only  in  the  presence  of  free 
oxygen.  Cultivation  at  high  temperatures  causes  some  organ- 
isms to  lose  the  power  of  pigment  production.  Some  pigments 
are  soluble  in  water,  others  in  alcohol,  and  others  in  ether  and 
various  fat  solvents.  They  are  of  little  economic  importance, 
but  are  of  value  to  the  systematic  bacteriologist  in  the  separation 
and  identification  of  species. 

LIGHT  PRODUCTION  BY  MICROORGANISMS 
Several  species  of  bacteria  and  fungi  are  known  that  give 


Fig.  26. — A  bacterial  lamp.  The  inner  wall  of  the  flask  is  coated  with  a 
medium  on  which  there  is  growing  Bacterium  phosphoreum.  Photographed  by 
its  own  light  (Molisch). 

off  light.     These   are  said  to  be  photogenic.     Bacteria  of  this 
type  are  found   commonly  in  the  water  of  the  ocean,  and  are 


58  VETERINARY   BACTERIOLOGY 

easily  isolated  from  salt  fish.  When  grown  in  a  test-tube,  they 
are  sometimes  sufficiently  luminous,  so  that  they  may  be  photo- 
graphed by  their  own  light. 

FERMENTATION  AND  ENZYME  PRODUCTION 

All  microorganisms  have  their  protoplasm  bounded  by  the 
ectoplast,  a  semipermeable  membrane,  as  previously  shown.  The 
cell  wall,  when  present,  seems  to  be  a  mechanical  protection  and 
support,  and  is  readily  permeable  to  most  substances  in  solution, 
so  may  be  disregarded  in  discussion.  Whatever  food  or  other  mate- 
rials are  taken  into  the  protoplasm  must  pass  by  diffusion  through 
the  ectoplast.  This  membrane,  on  the  other  hand,  must  prevent 
any  valuable  constituent  of  the  cell  from  leaving  by  diffusion. 
Its  action  is,  therefore,  selective.  Microorganisms  do  not  always 
find  the  potential  food  materials  with  which  they  are  surrounded 
suitable  for  food,  for  they  may  be  in  a  solid  form  or,  if  in  solution, 
of  such  a  character  that  they  cannot  pass  through  the  ectoplast. 
Many  organisms  find  it  necessary  to  so  change  this  food  and 
digest  it  that  it  may  be  assimilated.  Once  inside  the  cell,  it 
usually  is  not  of  such  a  character  that  it  can  be  built  up  directly 
into  the  protoplasm  and  further  changes  are  necessary;  or  if  the 
material  is  used  simply  as  a  source  of  energy  and  not  incorporated 
into  the  cell  substance,  it  is  essential  that  the  cell  have  some  means 
of  developing  this  energy.  All  cells  accomplish  these  changes  by 
means  of  enzymes  (Gr.  en,  within,  zyme,  leaven).  A  dis- 
tinction was  once  made  between  the  so-called  organized  and 
unorganized  ferments.  The  former  was  held  to  be  living  cells 
which  could  bring  about  a  change  or  fermentation,  the  latter  any 
cell  secretion  which  could  bring  about  such  changes.  In  other 
words,  jorgani/cd  ferments  were  supposed  to  owe  their  activity 
directly  to  the  protoplasm,  the  unorganized,  to  substances  secreted 
by  the  protoplasm.  This  distinction  is  no  longer  maintained, 
as  it  seems  altogether  probable  that  fermentative  changes  of 
whatever  kind  are  brought  about  by  the  secreted  enzymes  or 
unorganized  ferments.  Enzymes  may  be  intra-  or  extracellular. 
The  intracellular  enzymes  are  extracted  from  the  protoplasm 
with  difficulty,  and  during  the  life  of  the  cell  do  not  leave  it. 
Such  an  enzyme  is  that  of  bread  or  brewer's  yeast  (the  zymase), 


PHYSIOLOGY   OF   MICROORGANISMS  59 

which  converts  dextrose  into  alcohol  and  carbon  dioxid.  Extra- 
cellular enzymes  are  usually  digestive  in  their  action.  Different 
kinds  attack  different  substances.  Microorganisms  are  known 
which  produce  enzymes  that  will  break  down  cellulose,  starch, 
sugars,  fats,  and  proteins  into  simpler  substances.  The  action 
of  an  enzyme  is  said  to  be  specific;  a  given  enzyme  will  in  gen- 
eral change  only  one  type  of  material. 

Enzymes  are  said  to  be  organic  catalysts  (Gr.  kata,  down, 
lyo,  to  dissolve),  that  is,%  they  bring  about  changes  without 
themselves  becoming  part  of  the  final  product.  Many  inorganic 
catalysts,  such  as  finely  divided  platinum,  are  known  to  the 
chemist.  Although  catalysts  do  not  form  a  part  of  the  final 
products,  they  certainly  are  a  part  of  some  of  the  intermediary 
products  in  many  cases,  but  become  free  again  when  the  action  has 
been  completed.  Enzymes,  then,  are  peculiar  in  that  they  are 
not  used  up  in  the  using.  Theoretically  the  amount  of  change  that 
can  be  brought  about  by  a  given  enzyme  is  limited  only  by  the 
time  and  conditions  under  which  it  must  act. 

Most  enzymes  produce  changes  that  are  hydrolytic  in  nature, 
that  is,  they  bring  about  the  incorporation  of  water  into  the  organic 
molecule  with  resultant  disintegration.  The  digestion  of  gelatin 
by  the  bacterial  enzyme  gelatinase,  the  conversion  of  starch  into 
maltose*  by  ptyalin  and  diastase,  the  digestion  of  proteins  by 
pepsin,  the  conversion  of  saccharose  or  cane-sugar  into  invert 
sugar  by  invertase,  and  the  clotting  of  milk  by  rennet  are  a  few 
examples  of  such  hydrolytic  changes.  The  following  reaction 
illustrates  the  hydrolytic  cleavage  of  saccharose  by  the  invertase 
produced  by  yeast: 

C12H,2OU   +  H2O  +  Invertase  !z=;  C6H12O6  4-  C6H12O6  +   Invertase. 

Saccharose.  Dextrose.  Levulose. 

Other  enzymes  are  active  oxidizers.  Changes  of  color  in 
dead  or  injured  plant  and  animal  tissues  are  sometimes  due  to 
such  oxidases.  For  example,  potatoes  turn  black  and  apples 
become  brown  when  the  cells  are  bruised.  Some  other  enzymes 
are  said  to  be  splitting.  One  of  the  best  examples  of  these  is  the 
zymase  of  yeast,  which  converts  dextrose  into  alcohol  and  carbon 
dioxid. 


60  VETERINARY   BACTERIOLOGY 

C6H12O8  +  Zymase  =   2C2H5OH   +   2CO2   4-  Zymase. 

Dextrose.  Alcohol.  Carbon 

dioxid. 

Although  alcohol  and  carbon  dioxid  represent  the  end-products, 
it  is  by  no  means  certain  that  intermediate  hydrolytic  products 
are  not  formed,  and  this  splitting  action  may  be  essentially  hydro- 
lytic. Reducing  enzymes  have  also  been  demonstrated  in  plant 
and  animal  tissues  and  undoubtedly  occur  in  microorganisms. 

The  autolytic  (Gr.  auto,  self,  lyo,  dissolving)  enzymes  de- 
serve particular  mention.  Enzyme's  are  known  to  occur  in 
most  animal  and  plant  cells  that  will,  at  least  partially,  digest 
the  cells  in  which  they  occur.  The  rigor  mortis  or  stiffening  of  the 
tissues  of  an  animal  after  death  is  due  to  such  an  autolytic  enzyme 
which  coagulates  the  muscle  protoplasm.  The  softening  of  the 
tissues  which  occurs  later,  the  so-called  "  ripening  "  of  meat,  is  due 
in  part  to  the  action  of  another  proteolytic  enzyme  which  carries  the 
digestion  somewhat  further.  Microorganisms  contain  such  en- 
zymes, and  when  the  cells  die,  as  in  an  old  culture,  they  are  then 
partially  digested.  This  autolytic  action  we  shall  find  to  be  of 
some  practical  significance  in  a  discussion  of  disease  production 
and  immunity,  as  by  it  certain  poisonous  substances  may  be 
released  from  the  cell. 


CHAPTER  IV 

CHANGES  OF  ECONOMIC    SIGNIFICANCE  BROUGHT  ABOUT  BY 
NON-PATHOGENIC  ORGANISMS 

MICROORGANISMS  bring  about  many  changes,  both  analytic 
and  synthetic,  in  the  media  in  which  they  are  cultivated.  In 
any  such  medium  growth  products  of  many  kinds  will  be  found. 
These  fermentative  products  may  originate  from  the  activity  of 
extracellular  enzymes,  may  consist  of  substances  excreted  from 
the  cell  as  the  product  of  intracellular  enzymes,  or  as  a  result 
of  the  metabolic  activity  of  the  cell;  they  may  be  products  of  syn- 
thetic action  (as  the  slimes  and  gums  produced  by  a  solution  of 
the  bacterial  capsule),  or  they  may  be  substances  produced  by  the 
autolytic  activity  of  intracellular  enzymes  after  the  death  of  the 
cell.  Naturally,  the  products  will  vary  greatly  with  the  species 
of  organism,  the  medium  in  which  it  is  grown,  and  the  character 
of  the  physical  environment. 

From  the  standpoint  of  the  veterinarian,  microorganisms 
are  of  most  importance  because  many  of  them  produce  disease. 
It  would,  however,  give  a  false  impression  of  the  place  and  function 
of  microorganisms  in  nature  to  neglect  at  least  a  brief  consid- 
eration of  some  of  the  other  changes  which  they  can  bring  about. 
In  this  chapter  some  of  the  more  important  will  be  considered. 

The  well-nigh  universal  distribution  of  bacteria  and  other 
microorganisms  should  be  emphasized.  They  are  to  be  found  in 
the  soil  in  great  numbers,  rich  surface  soil  containing  from  100,000 
to  5,000,000  bacteria  to  every  dry  gram.  Their  dried  bodies  and 
spores  are  constantly  present  free  or  attached  to  dust  particles  in 
the  air.  They  are  to  be  found  in  all  surface  waters  in  considerable 
numbers  and  are  present  even  in  the  water  from  deep  wells. 
They  grow  upon  the  surface  of  the  skin  of  animals,  and  the  mouth 
and  digestive  tract  support  a  large  and  varied  flora.  It  is  apparent 
that  whenever  conditions  are  favorable  for  the  growth  of  micro- 
organisms they  will  be  present  to  begin  growth. 

61 


62  VETERINARY  BACTERIOLOGY 

The  changes  brought  about  by  bacteria  in  nature  are  of  such 
importance  that  but  for  their  continuance  plant  and  animal 
life  on  earth  would  quickly  cease  to  exist.  The  fertility  of  the 
soil  and  the  consequent  production  of  all  food  stuffs  is  directly 
due  to  certain  of  the  microorganisms  present. 

Production  of  Alcohol. — The  various  alcohols,  but  more  par- 
ticularly ethyl  alcohol,  are  produced  by  certain  bacteria,  yeasts, 
and  molds.  It  has  been  shown  that  in  the  yeast  the  ability  to 
bring  about  this  change  is  resident  in  the  intracellular  enzyme, 
zymase.  Probably  similar  enzymes  are  present  in  the  alcohol- 
producing  bacteria  and  molds.  The  common  bread  or  brewer's 


Fig.  27. — Brewer's  yeast,  Saccharomyces  cerevisice. 

yeast  is  the  form  most  commonly  used  in  the  manufacture  of 
alcoholic  beverages,  but  certain  molds  have  been  found  very 
useful  in  the  production  of  alcohol  for  industrial  purposes.  Alco- 
hol is  commonly  produced  by  the  fermentation  of  one  of  the 
hexose  monosaccharids,  such  as  dextrose.  The  reaction  may  be 
given  as  follows: 

C6H12O9  =  2C2H5OH   +  2CO2. 

Dextrose.  Alcohol.  Carbon 

dioxid. 

Yeast  is  utilized  for  its  other  product  of  fermentation,  carbon 
dioxid,  by  the  baker  in  bread  making.  Higher  alcohols,  such  as 
butylic  and  amylic,  are  also  formed.  The  yeasts  which  produce 
this  change  are  quite  widely  distributed  in  nature,  being  par- 
ticularly abundant  on  the  surface  of  fruits  and  in  saccharine 
liquids.  Fruit  juices  and  other  solutions  containing  sugar  if 
allowed  to  stand,  therefore  undergo  "  spontaneous  "  fermenta- 
tion, with  production  of  ciders,  wines,  and  similar  beverages. 

Production  of  Acids. — Several  of  the  organic  acids  are  com- 
monly produced  by  fermentative  organisms.     Three  of  these  are 


CHANGES  BROUGHT  ABOUT  BY  NON-PATHOGENIC  ORGANISMS  63 

of  particular  economic  importance,  namely  lactic,  acetic,  and 
butyric.  A  great  variety  of  others  are  occasionally  produced, 
usually  in  small  quantities  only. 


B 

Fig.  28. — Lactic  acid  bacteria:  A,  Streptococcus  lacticus;  B,  Bacillus  bulgaricus. 

Lactic  Acid. — Dextrose  and  some  other  monosaccharids  are 
converted  into  lactic  acid  by  several  common  organisms.  The 
reaction  may  be  empirically  represented  as  follows: 

C6H12O6  =  2C2H/OH-COOH. 

Dextrose.  Lactic  acid. 

The  reaction  occurs  most  frequently  in  milk  which  is  allowed  to 
stand.  In  this  case  the  lactose  or  milk-sugar  is  first  broken 
down  into  the  monosaccharids  before  being  converted  into  lactic 

acid. 

CI2H220U  +  H20  =  C6H1208  +  C6H1206. 

Lactose.  Dextrose.  Galactose. 

The  formation  of  lactic  acid  in  milk  is  of  the  greatest  economic 
importance,  as  the  organisms  which  produce  this  acid  are  the  ones 


Fig.  29. — Acetic  acid  organism,  Bacillus  aceti:  a,  Normal  individuals;  b,  in- 
volution forms.     (Adapted  from  Hansen.) 

which  are  necessary  to  the  development  of  proper  flavors  and 
quality  in  butter  and  cheese.  This  acid  is  also  produced  in 
the  manufacture  of  sauer-kraut  and  to  some  extent  in  silage. 
The  lactic  acid  formed  in  milk  is  instrumental  in  preventing  the 
growth  of  putrefactive  and  other  undesirable  bacteria. 


64  VETERINARY   BACTERIOLOGY 

Acetic  Acid. — Acetic  acid  is  the  most  important  and  the 
characteristic  constituent  of  vinegar.  It  is  produced  by  several 
species  of  bacteria  by  the  oxidation  of  ethyl  alcohol  according 
to  the  following  reaction : 

C2H5OH   +  02  =   CH3COOH   +  H2O. 

Alcohol.  Acetic  acid. 

Any  solution  containing  alcohol,  if  left  in  contact  with  free  oxygen, 
will  commonly  undergo  this  fermentation  spontaneously,  as 
Bacillus  acetiy  the  organism  usually  responsible,  is  ubiquitous. 
To  insure  rapid  and  efficient  fermentation  the  cider  or  other 
alcoholic  solution  is  sometimes  inoculated  with  mother  of  vinegar, 
a  mass  of  the  organism  which  commonly  forms  a  mat  upon  the 
surface  of  the  fermenting  liquid. 


Fig.  30. — Butyric  acid  bacteria,  Bacillus  bulyricus.     (Adapted  from  Fischer.) 

Butyric  Acid. — Under  anaerobic  conditions  saccharine  solutions 
are  apt  to  undergo  butyric  acid  fermentation  as  a  result  of  the 
development  of  the  Bacillus  butyricus  or  a  related  form.  The 
reaction  may  be  represented  as  follows: 

C6H1206  =  C3H7COOH  +  2C02   +  2H2. 

Dextrose.  Butyric  acid. 

Butyric  acid  has  an  exceedingly  disagreeable  odor  and  taste, 
hence  the  growth  of  this  organism  in  any  saccharine  or  starchy 
food  substance  renders  it  unfit  for  use.  Inasmuch  as  these 
organisms  are  all  spore  producers,  they  resist  heat  well,  and  as 
they  are  anaerobic  they  will  grow  when  sealed  in  a  can  and  all 
air  excluded.  Rancidity  in  butter  is  sometimes  due,  in  part 
at  least,  to  the  development  of  butyric  acid. 

Decay  and  Putrefaction. — A  distinction  is  sometimes  made 
between  the  terms  decay  and  putrefaction.  The  former  is  said  to 


CHANGES  BROUGHT  ABOUT  BY  NON-PATHOGENIC  ORGANISMS  65 

be  decomposition  of  organic  matter  brought  about  by  the  aerobic 
bacteria,  the  latter  by  the  anaerobic  types.  This  distinction 
is  not  always  acknowledged  and  adhered  to,  however.  The 
substances  produced  by  the  decomposition  by  bacteria  depend,  of 
course,  quite  largely  upon  the  nature  of  the  material  to  be  de- 
composed. The  carbohydrates  and  fats  break  down  into  alcohols. 

,  but  the  proteins  are  split  into  a  great 


variety  of  substances.  Other  agents,  as  acids  and  alkalis,  will 
break  up  the  proteins  in  a  similar  manner  and  into  many  of  the 
same  substances  as  do  bacteria.  Chemists  have  in  recent  years 
demonstrated  that  proteins  are  made  up  of  large  numbers  of 
molecules  of  the  a-amino  acids  linked  together.  An  oc-amino  acid 
is  an  organic  acid  that  has  the  NH2  group  in  the  alpha  position, 
that  is,  next  the  carboxyl.  For  example,  the  amino  acid  corre- 
sponding to  C2H5CH2COOH,  butyric  acid,  is  C2H5CHNH2COOH. 
When  these  constituent  links  of  the  protein  molecule  are  forced 
apart,  they  usually  appear  in  the  form  of  one  of  about  twenty  com- 
pounds which  have  been  grouped  as  primary  protein  derivatives. 
Some  of  these  normal  derivatives  are  further  altered  by  bacteria. 
Among  them  have  been  found  certain  compounds  called  ptomains, 
some  of  which  are  known  to  be  very  poisonous.  The  splitting 
usually  continues  until  much  of  the  organic  matter  is  reduced 
to  comparatively  simple  compounds,  such  as  H2S,  CO2,  CH4,  and 
NH3.  The  process  ofjprotein  disintegration  is  called  proteolysis. 
It  usually  occurs  in  several  distinct  stages.  The  proteins  are 
first  broken  down  into  relatively  complex  substances  called  prote- 
oses,  these  then  are  broken  down  into  peptones.  This  is  called 
peptonization,  as  it  is  essentially  the  change  that  may  be  brought 
about  by  the  enzyme  pepsin  from  the  stomach.  The  process  con- 
tinues and  the  peptones  become  amino  acids  and  at  last  ammonia 
is  liberated.  From  an  economic  point  of  view  this  liberation  of 
ammonia  has  the  greatest  significance,  for  from  this  transformation 
comes  all  the  nitrogenous  material  used  by  plants  and  indirectly 
by  animals  as  food.  This  is  the  essential  transformation  that  all 
organic  nitrogenous  fertilizers,  such  as  barnyard  manure  and 
dried  blood,  will  undergo  before  they  can  be  of  any  use  to  higher 
plants.  By  such  changes  water  contaminated  by  sewage  purifies 
itself. 

5 


66  VETERINARY   BACTERIOLOGY 

Some  of  the  nitrogenous  products  of  bacterial  decomposition 
are  worthy  of  note,  inasmuch  as  they  are  used  in  the  laboratory  in 
the  differentiation  of  certain  species.  The  most  important  of 
these  are  indol  and  skatol.  They  are  organic  compounds  having 

the  following  formulas: 

/CH3 

/V^.H.,X  /      \*>     x\ 

CflH4  CH  C6H4<  CH. 


Indol.  Skatol. 


Indol  is  produced  by  certain  bacteria  when  growing  in  a  solution 
of  peptone.  It  is  identified  by  the  addition  of  nitrous  acid,  with 
which  it  combines  to  form  nitroso  indol,  a  bright  red  compound. 
In  making  the  test  in  the  laboratory  it  is  customary  to  add  a 


t 

Fig.  31. — Some  decay-producing  and  putrefactive  bacteria. 

few  drops  of  concentrated  sulphuric  acid,  followed  by  a  dilute  solu- 
tion of  nitrite.  The  sulphuric  acid  breaks  up  the  nitrite,  with  the 
formation  of  free  nitrous  acid,  which  then  unites  with  the  indol. 
Indol  and  skatol  are  also  formed  in  the  intestines  by  the  activity 
of  certain  of  the  bacteria  found  there  and  are  of  considerable 
physiological  significance. 

Reduction  Processes  in  Inorganic  Compounds. — Changes  similar 
to  those  just  discussed  are  sometimes  brought  about  by  bacteria 
in  inorganic  compounds.  When  nitrates  are  in  solution  together 
with  organic  substances  and  under  anaerobic  conditions,  the 
bacteria  present  in  many  cases  will  reduce  the  nitrates  to  nitrites 
and  the  nitrites  to  free  nitrogen,  apparently  in  order  to  utilize 
the  oxygen.  This  process  is  usually  called  denitrification  because 
the  medium  loses  nitrogen,  but  is  more  correctly  a  reduction  or 
deoxidation.  Sulphates  are  reduced  to  sulphites  and  even  to 
sulphids  under  similar  conditions.  For  example,  the  sewage  from 


CHANGES  BROUGHT  ABOUT  BY  NON-PATHOGENIC  ORGANISMS  67 

a  city  whose  water  supply  contains  a  large  percentage  of  sulphates 
will  develop  hydrogen  sulphid  in  considerable  quantities  if  it  is 
put  under  anaerobic  conditions.  Other  reductions  of  a  similar 
nature  have  been  described  for  chlorates. 


Fig.  32. — Denitrifying  bacteria:  A,  .Boct'ZZus  coli,  which  changes  nitrates  into 
nitrites;  B,  Bacillus  denitrificans,  which  produces  free  nitrogen  from  nitrates. 

Oxidation  of  Inorganic  Compounds. — Bacteria  and  other 
microorganisms  that  live  in  the  presence  of  oxygen  are  sometimes 
active  oxidizers  of  inorganic  compounds,  securing  in  this  manner 
the  energy  that  is  necessary  for  their  various  growth  processes. 


Fig.  33. — Sulphate  reducing  spirillum,  Spirillum  desulfuricans. 

Oxidation  of  Hydrogen  Sulphid. — Waters  containing  hydrogen 
sulphid,  as  do  many  of  the  so-called  mineral  springs,  usually 
contain  bacteria  which  gain  their  energy  for  food  manufacture 


Fig.  34. — Microorganisms  that  oxidize  hydrogen  sulphid:    A,  B,  Beggiatoa 
sp.;  C,  Thiophysa  volutans  (Hinze). 

and  growth  from  the  oxidation  of  this  substance.  The  slimy 
black  and  white  deposit  commonly  found  in  such  waters,  when 
examined  microscopically,  will  be  seen  to  be  made  up  of  masses  of 


68  VETERINARY   BACTERIOLOGY 

Beggiatoa  and  similar  organisms  whose  cells  will  be  found  packed 
with  sulphur  granules.  Probably  the  following  reaction  accounts 
for  this  formation  of  free  sulphur: 

2H2S  +  O2  =  2H2O  +  S2. 

The  process  is  carried  still  farther  if  there  is  any  deficiency  of 
the  hydrogen  sulphid,  and  the  free  sulphur  is  converted  into 
sulphuric  acid  and  sulphates. 

S2  4-  3O2  =  2SO3 
H2O   +  SO3  =   H2SO4. 

The  sulphuric  acid  is,  of  course,  at  once  neutralized  by  the  bases 
present  in  the  water. 

Oxidation  of  Iron. — Many  natural  waters  contain  ferrous  car- 
bonate or  some  similar  salt  of  iron.     Certain  bacteria  oxidize 


Fig.  35. — Microorganisms  that  oxidize  ferrous  to  ferric  iron:  a,  Lep- 
tothrix  ochracea;  b,  Gallionella  ferruginea;  c,  Spirophyllum  ferrugineum . 
(Adapted  from  Ellis.) 

this  to  ferric  hydrate,  and  deposit  this  insoluble  material  in 
their  sheaths.  The  reaction  may  be  represented  as  follows: 

2Fe2C03  +  3H20  4-  O  =  Fej(OH)6  4-  2CO2. 

Probably  these  organisms  make  use  of  the  energy  obtained  by 
this  reaction  in  the  same  manner  that  the  sulphur  bacteria  do  the 
oxidation  of  the  sulphur,  to  secure  energy  for  the  formation  of  their 
foods  and  to  gain  the  energy  needed  for  growth  ;m<l  development. 
These  organisms  are  particularly  apt  to  occur  in  well  water  or 
spring  water  laden  with  iron,  and  have  in  some  cases  caused 
considerable  trouble  by  clogging  the  water  pipes  with  1  heir  growth. 
It  is  known  that  the  bog  iron  ore  of  Sweden  and  probably  the 


CHANGES  BROUGHT  ABOUT  BY  XOX-PATHOGEXIC  ORGANISMS  69 

great  iron  beds  of  northern  Minnesota  have  been  deposited  by 
the  activity  of  such  organisms. 

Oxidation  of  Ammonia. — In  most  soils  there  are  numerous 
bacteria  that  oxidize  free  ammonia  to  nitrous  acid,  and  by  neutral- 
ization with  the  soil  bases  form  nitrites.  These  organisms  do  not 
develop  well  in  the  laboratory  in  the  presence  of  organic  matter. 
It  seems  evident  that  they  utilize  the  energy  secured  from  the 


Fig.  36. — Bacteria  that  oxidize  ammonia  and  nitrous  acid   to   nitrous 
and  nitric    acid  respectively:    a,   Xitrosomonas  europea;    b,  N.  javet 
Xitrobacter  (Winogradsky) . 

oxidation  of  the  ammonia  to  build  up  their  protoplasm  out  of 
simple  materials.  They  are  among  the  best  examples  of  the 
strictly  prototrophic  bacteria.  Organisms  capable  of  bringing 
about  this  change  are  called  nitroso-bacteria.  The  reaction  may  be 
represented  as  follows: 

XH,  +  202  =  HX03  +  H20. 

This  is  the  first  of  the  two  steps  in  the  process  called  nitrification 
in  the  soil. 

Oxidation  of  Nitrous  Acid. — The  nitrous  acid  formed  in  the 
soil  and  in  water,  etc.,  by  the  preceding  group  is  further  changed 
by  another  group  of  organisms  called  the  nitrate  bacteria.  Like 
the  preceding,  they  are  widely  distributed  in  water  and  soil, 
and  complete  the  process  called  nitrification  or,  better,  oxidation 
of  nitrogen.  The  nitrates  produced  by  their  activity  are  the 
source  of  nitrogen  for  green  plants.  A  few  of  the  latter  are  able 
to  make  use  of  nitrogen  in  the  form  of  ammonia  compounds,  but 
in  nature  this  rarely  occurs.  The  reaction  may  be  represented  as 
follows : 

2HNO2   4-  O2   -  2HXO3. 

It  is  probable  that  the  fertility  of  the  average  soil  is  more  largely 
determined  by  the  maintenance  of  conditions  favorable  to  the 


70  VETERINARY   BACTERIOLOGY 

development  of  these  nitrifying  organisms  than  by  any  other 
single  factor.  Their  importance  in  nature  as  essentials  for  the 
growth  of  the  higher  plants  and,  therefore,  of  animals  can  scarcely 
be  overestimated. 

Nitrogen  Fixation. — The  free  nitrogen  of  the  air  is  so  inert 
that  very  few  living  plants  are  capable  of  making  use  of  it  in 
the  building  up  of  their  bodies.  None  of  the  green  plants  can 
bring  this  about  of  themselves.  Certain  molds  and  bacteria 
are  able  to  make  use  of  this  source  of  nitrogen,  however,  and 
are,  therefore,  of  the  greatest  economic  importance.  The  fer- 
tility of  the  soil  is  largely  dependent  upon  the  fixed  nitrogen  that 
it  contains,  and  the  taking  up  of  the  free  nitrogen  by  these 
organisms  ultimately  renders  it  available  to  other  forms  of  plants. 
This  does  not  mean  that  the  bacteria  take  up  the  nitrogen  from  the 


A. 

Fig.  37. — Free  living  or  non-symbiotic  nitrogen-fixing  bacteria:  A,  Bacillus 
(Clostridium}  pastorianum;  B,  Azotobacter;  a,  A.  chroococcum;  b,  A.  agilis. 
(A  after  Winogradsky,  B  after  Beyerinck.) 

air  and  immediately  transform  it  into  nitrates  for  the  use  of  the 
higher  plants,  but  that  it  is  built  up  into  their  protoplasm  and 
ultimately  is  set  free  by  the  death  and  decomposition  of  the  organ- 
isms. Microorganisms  which  make  use  of  free  nitrogen  commonly 
utilize  carbonaceous  materials  as  a  source  of  energy. 

Organisms  capable  of  fixing  nitrogen  are  subdivided  into  two 
general  groups,  those  which  live  free  in  soils  and  those  which  live 
in  or  on  the  roots  of  certain  plants  in  a  kind  of  symbiosis.  The 
free  living  organisms  which  fix  nitrogen  belong  to  three  general 
groups:  first,  certain  anaerobic  types  belonging  to  the  general 
group  of  butyric  acid  bacteria;  second,  certain  aerobic  species; 
third,  a  few  molds.  The  anaerobic  organism  known  to  fix  the 
nitrogen  is  Bacillus  (Clostridium)  pastorianum.  Probably  this 
organism  is  not  of  the  greatest  importance,  as  the  conditions  for 


CHANGES  BROUGHT  ABOUT  BY  NON-PATHOGENIC  ORGANISMS  71 

its  development  do  not  often  obtain.  Bacteria  of  the  nitrogen- 
fixing  aerobic  type  belong  to  the  group  called  Azotobacter.  These 
organisms  are  abundant  in  many  soils  and  fix  considerable  quan- 
tities of  nitrogen,  gaining  energy  therefor  by  oxidizing  the  car- 
bonaceous materials  from  dead  plant  tissues.  The  addition  of 
straw,  for  example,  to  a  soil  will  furnish  sufficient  food  so  that 
these  bacteria  will  bring  about  an  appreciable  increase  in  the 
nitrogen  content.  The  importance  of  the  molds  in  this  connection 
is  not  fully  understood,  but  several  species  have  been  described 
which  are  capable  of  fixing  nitrogen. 

The  microorganisms  which  fix  nitrogen  in  symbiosis  with 
higher  plants  may  be  divided  into  two  groups,  those  bacteria 
which  grow  upon  the  roots  of  legumes  and  the  molds  which 
grow  on  the  roots  of  certain  other  plants.  All  plants  belonging 


Fig.  38. — Bacillus  radicicola:  a,  Normal  bacillar  form;  6,  bacteroids  or  involu- 
tion forms. 

to  the  legume  or  pulse  family,  such  as  clover,  alfalfa,  peas,  beans, 
etc.,  usually  bear  upon  their  roots  tubercles  or  nodules  which, 
when  opened,  are  found  to  be  made  up  of  cells  tightly  packed  with 
bacteria.  It  has  been  shown  experimentally  that  these  organisms 
growing  within  the  roots  in  some  way  take  up  free  nitrogen  from 
the  air  and  eventually  turn  it  over  to  the  host  plant,  so  that  the 
legumes  are  not  dependent  for  their  development  upon  nitrogen 
which  may  be  present  in  the  soil,  but  can  make  use  of  the 
free  nitrogen  of  the  air  as  well.  These  plants  are,  therefore, 
very  important  in  agriculture  in  the  maintenance  and  increase  of 
soil  fertility.  This  organism,  known  as  Bacillus  radicicola  (Fig. 
38),  enters  the  young  growing  root  through  a  root  hair  and  causes 
a  kind  of  tumor  formation  in  the  tissues  of  the  root,  resulting  in 
the  development  of  the  nodule.  The  organism  is  at  first  a  straight 
rod,  but  later,  when  growing  inside  the  cells  of  the  host,  it  becomes 


72 


VETERINARY   BACTERIOLOGY 


much  enlarged  and  shows  many  involution  forms.  The  other 
organisms  growing  symbiotically  within  or  upon  the  roots  of 
plants  are  all  molds.  They  develop  either  upon  the  surface 
of  the  root,  forming  a  white,  cottony,  floccose  covering,  or  they 
grow  in  the  tissues  just  below  the  epidermis.  They  sometimes 
cause  nodules  to  develop,  as  is  the  case  with  the  Russian  olive  and 
the  alder,  or  they  produce  no  characteristic  overgrowth  of  tissues 


Fig.  39. — Nodules  on  the  root  of  a  legume,  Soy  bean.     (Moore,  U.  S.  Dept. 

Agr.) 

at  any  one  point,  but  are  found  quite  uniformly  present  upon  the 
young  growing  roots.  Certain  trees,  such  as  the  oak  and  the 
pine,  particularly,  when  growing  in  nitrogen  poor  soils,  show  the 
development  of  this  mycorrhiza  (Gr.  fungus  and  root).  It  has 
been  shown  that  these  molds  are  quite  active  in  taking  up  free 
nitrogen  from  the  air  and  are  of  benefit  to  the  plant  upon  which 
they  occur. 


CHANGES  BROUGHT  ABOUT  BY  NON-PATHOGENIC  ORGANISMS  73 

The  Nitrogen  Cycle. — The  relationship  of  microorganisms  to 
nitrogen  and  its  compounds  has  been  noted  in  the  preceding  pages. 
These  changes  may  be  summarized  as  follows:  Certain  bacteria 
break  down  organic  compounds  containing  nitrogen  with  the  ulti- 
mate liberation  of  ammonia.  Other  species  change  the  ammonia 
to  nitrous  acid  and  nitrites.  Still  other  species  transform  the 
nitrites  to  nitrates,  and  these  the  higher  plants  take  up  from  the 
soil  and  transform  again  into  complex  organic  substances.  These 
eventually  again  decay  or  are  eaten  by  animals  and  converted  into 
"animal  tissues.  The  nitrogen  of  both  plant  and  animal  tissues 


OnimoJ 


Qm*ion  i  TI  car/on 


Fig.  40. — Nitrogen  cycle.     Changes  brought  about  by  bacteria  indicated  by 
solid  lines,  other  changes  by  dotted  lines. 


ultimately  undergoes  the  change  first  noted  and  the  nitrogen  again 
appears  as  ammonia.  This  series  of  changes  is  called  the  nitrogen 
cycle.  It  is  to  be  noted  in  addition  that  some  bacteria  are  found 
which  decompose  nitrates  with  the  formation  of  nitrites  and 
liberate  free  nitrogen  in  the  so-called  process  of  denitrification. 
Other  species,  either  alone  or  in  symbiosis  with  higher  plants, 
take  up  and  fix  free  nitrogen  from  the  air  and  eventually  convert  it 
into  a  form  available  for  higher  plants.  These  changes  may  be 
better  understood  by  reference  to  the  accompanying  diagram. 


74  VETERINARY  BACTERIOLOGY 

Miscellaneous  Changes. — Many  changes  are  brought  about 
by  bacteria  other  than  those  which  are  here  discussed.  Micro- 
organisms are  of  importance  in  tanning,  curing  of  tobacco,  the 
preservation  of  food-stuffs,  such  as  silage  and  sauer-kraut,  the 
retting  of  flax  and  hemp,  the  curing  of  the  so-called  burnt  or 
heated  hay,  and  in  many  other  ways.  It  is  to  be  remembered  that 
probably  in  all  cases  these  changes  are  brought  about  by  the 
enzymes  produced  by  the  bacteria. 


CHAPTER  V 

CLASSIFICATION  OF  MICROORGANISMS 

IT  is  necessary  in  a  consideration  of  organisms  belonging  to 
either  the  plant  or  animal  kingdoms  to  divide  or  separate  them 
into  groups,  with  their  apparent  relationships  as  the  basis  for  the 
grouping.  Microorganisms  of  pathogenic  significance  we  have 
previously  divided  into  the  four  groups — bacteria,  yeasts,  molds, 
and  protozoa.  A  discussion  of  the  classification  of  the  last  will 
be  reserved  to  the  chapter  on  Diseases  Produced  by  Protozoa. 

The  classification  of  micro-organisms  is  by  no  means  in  a  satis- 
factory state.  Many  bacteriologists  and  others  who  have  inves- 
tigated diseases  have  failed  to  recognize  the  importance  of  simple 
classifications  and  have  introduced  many  new  names  needlessly. 
It  is  a  principle  of  nomenclature  accepted  since  the  time  of  Lin- 
naeus that  every  plant  and  animal  belonging  to  a  distinct  type  or 
species  shall  receive  a  Latin  name,  this  name  to  be  made  up  of  two 
words  only.  The  second  of  these  words  is  the  species  name,  and 
is  peculiar  to  the  particular  kind  under  consideration,  the  first  is 
called  the  genus  or  generic  name.  For  example,  among  higher  plants 
we  have  the  genus  Quercus,  or  oak,  which  is  subdivided  into  many 
species,  such  as  white  oak,  red  oak,  swamp  oak,  etc.  (Quercus  alba, 
rubra,  etc.) .  The  generic  name  is  applied  to  all  those  species  which 
resemble  each  other,  as  do  all  of  the  oaks.  Species  of  plants  and  ani- 
mals are  given  names  which  are  understood  to  serve  as  convenient 
terms  for  their  designation.  It  is  an  established  principle  that  the 
name  first  given  to  a  plant  or  an  animal  is  the  one  which  should 
always  be  used  whenever  that  name  is  in  accordance  with  certain 
rules.  Some  bacteriologists  have  made  the  mistake  of  believing 
that  a  scientific  name  should  be  a  description  or  even  a  descriptive 
term.  It  is  no  more  necessary  that  the  species  name  of  a  bacterium 
should  describe  that  bacterium  than  that  the  given  or  Christian 
name  of  an  individual  should  describe  him.  Disregard  of  this  rule 
has  resulted  in  some  very  unwieldy  names  being  given  to  micro- 

75 


76  VETERINARY   BACTERIOLOGY 

organisms,  for  example,  names  such  as  the  following  have  been 
applied  to  bacteria:  Bacillus  membranaceous  amethystinus  mobilis, 
Bacillus  argenteus  phosphorescens  liquefaciens,  and  even  the  fol- 
lowing, Bacillus  saccharobutyricus  fluorescens  liquefaciens  im- 
mobilis.  Such  names  are  given  under  the  mistaken  idea  that 
the  specific  name  should  be  a  description  of  the  species.  This  is 
not  customary  in  naming  any  of  the  higher  plants  and  animals, 
and  it  is  certainly  not  more  desirable  in  bacteria.  The  yeasts, 
molds,  and  protozoa  have  more  commonly  been  studied  by  those 
who  have  had  technical  training  in  nomenclature  than  have  the 
bacteria,  consequently  the  classification  of  these  forms  is  on  a 
much  more  satisfactory  basis.  The  only  justification  for  a  specific 
name  made  up  of  more  than  two  words  is  that  the  two  words 
taken  together  express  but  a  single  idea. 

CLASSIFICATION  OF  BACTERIA 

Many  different  classifications  have  been  proposed  for  bacteria, 
but  not  one  of  these  has  come  into  general  use.  A  careful  exam- 
ination of  different  texts  in  bacteriology,  particularly  those 
devoted  to  the  pathogenic  bacteria,  will  show  that  different  sys- 
tems and  schemes  of  classifications  are  used  in  dealing  with 
closely  related  organisms.  Not  only  have  the  groups  been  fre- 
quently changed,  but  many  different  names  have  been  applied 
to  almost  every  one  of  the  pathogenic  bacteria.  The  consequence 
is  that  in  studying  any  pathogenic  organism  it  is  necessary  to 
give  not  only  the  name  preferred  by  the  author  but  also  a  list 
of  the  synonyms  which  have  been  used  by  others.  It  seems 
probable  that  a  satisfactory  system  of  nomenclature  is  yet  to  be 
devised.  The  system  of  bacterial  classification  which  has  been 
most  generally  adopted  and  has  given  the  best  general  satisfaction 
is  that  of  Migula,  published  in  Engler  and  Prantl's  Synopsis  of 
Plant  Genera.  This  classification  has  been  somewhat  modified 
by  Frost  and  McCampbell  and  their  regrouping  is  perhaps  more  in 
accord  with  the  facts.  It  is  perhaps  more  important  that  a  dis- 
tinct and  satisfactory  classification  should  be  adhered  to  than 
that  a  single  classification  should  be  used  for  all  purposes. 
The  following  classification  is  the  one  which  will  be  adopted  in 
this  text.  It  is  based  upon  Frost  and  McCampbell's  modifica- 


CLASSIFICATION   OF   MICROORGANISMS  77 

tion  of  Migula's  scheme,  but  with  some  changes  which  seem  to 
coordinate  it  better  with  practice.  A  key  to  the  various  groups 
and  genera  of  the  bacteria  as  here  used  will  first  be  given,  fol- 
lowed by  a  brief  discussion  of  the  characteristics  of  the  more 
important  genera.  All  of  those  groups  of  bacteria  which  are 
not  of  economic  importance  or  which  have  no  pathogenic  members 
have  been  eliminated.  This  removes  some  fifteen  or  eighteen 
genera  which  have  no  common  or  economic  representatives  and 
are  chiefly  of  systematic  or  botanical  interest. 

KEY  TO  THE  GROUPS  AND  GENERA  OF  BACTERIA 

I.  Order  Eubacteria,  or  true  bacteria.     Cells  free  from  sulphur. 
Family  I.     Coccaceae.     Bacterial  cells  globular  when  in  a  free  state. 

Xon-motile. 

Cell  division  occurring  in  parallel  planes  resulting  in  the  formation  of 

chains Streptococcus. 

Cells  dividing  in  two  planes,  forming  plates  of  cells,  or  not  remaining 
united,  or  dividing  irregularly  to  form  irregular  masses.  .Micrococcus. 
Cell  division  occurring  in  three  planes,  all  at  right  angles,  the  cells  re- 
maining united  after  division  and  forming  cubes  or  packets.  .Sarcina. 
Motile. 

Same  as  Micrococcus,  but  with  organs  of  motion Planococcus. 

Same  as  Sarcina,  but  with  organs  of  motion Planosarcina. 

Family  II.     Bacteriaceae.     Cells  cylindric  in  shape  and  not  bent .  .  Bacillus. 

Family  III.     Spirillaceae.     Cells  in  the   form   of   corkscrews  or  spirals,  or 

segments  of  a  spiral.     Cells  fairly  rigid,  usually  motile  by  means  of  a 

flagellum  or  tuft  of  flagella  at  the  end Spirillum. 

Cells    forming   long,    thin,    and    tenuous    spirals.     Flagella,    if  present, 
demonstrated  only  with  difficulty.     Probably  protozoa  in  part  and 

not  true  bacteria Spirochceta. 

Family  IV.     Chlamydobacteriaceae.     Cells    cylindric,    united   in    threads, 
and  surrounded  by  a  sheath.     Arthrospores  sometimes  present. 

Filaments  show  no  branching Leptothrix. 

Filaments  show  false  branching Cladothrix. 

Filaments  show  true  branching. 

Arthrospores  or  conidia  produced Xocardia. 

;>ores  observed Actinomyccs. 

II.  Order  Thiobacteria.     Cells  containing  sulphur  granules. 

Filaments   surrounded  by  a  sheath Thiothrix. 

Filaments  not  surrounded  by  a  sheath Beggiatoa. 

Streptococcus. — The  term  Streptococcus  is  applied  to  any 
spherical  organism  whose  cells  occur  in  chains.  The  method  of 
development  has  already  been  discussed.  Spores  are  not  developed. 
In  some  forms  the  chains  break  up  readily  into  pairs,  to  which 


78  VETERINARY  BACTERIOLOGY 

the  name  Diplococcus  is  sometimes  applied.     This  has  been  used 
by  some  writers  as  a  genus  name. 

Micrococcus.  —  All  spherical  organisms  the  cells  of  which  do 
not  occur  either  in  chains  or  packets  are  generally  included  under 
the  genus  name  Micrococcus.  As  defined  by  Migula,  the  name 
strictly  applies  only  to  those  organisms  which  divide  alternately 
in  two  planes  at  right  angles  to  each  other,  forming  pairs  and 


Fig.  41.  —  Types  of  Streptococci:  a,  d,  Streptococci  consisting  of  uniform  ele- 
ments; 6,  Streptococcus  consisting  of  diplococcus  elements;  c,  Diplococcus. 

fours  and.  eventually  a  plate.  Very  few  bacteria  develop  in  this 
manner.  It  has  been  assumed  by  some  authors  that  division 
may  occur  in  the  two  planes,  but  the  cells  may  divide  at  such 
irregular  intervals  that  irregular  masses  of  the  organisms  may 
be  formed.  It  is  altogether  probable  that  some  of  the  cocci 
divide  irregularly  and  not  in  planes  strictly  perpendicular  or 
parallel  to  previous  planes  of  division.  This  results  in  the  forma- 


ooof 


Fig.  42.  —  Types  of  Micrococcus:  a,  Micrococcus  of  isolated  cells;  6,  Micro- 
coccus  showing  tetrads,  forming  plates  of  cells  or  merismopedia;  c,  Micrococcus 
with  cells  in  an  irregular  mass  —  Staphylococcus. 

tion  of  irregular  groups.  When  the  organisms  remain  united,  as 
frequently  occurs,  they  form  irregular  bunches,  to  which  the  name 
Staphylococcus  is  sometimes  given.  As  used  in  the  following  chap- 
ters, the  term  Staphylococcus  is  synonymous  with  Micrococcus. 

Bacillus.  —  As  used  here,  the  term  Bacillus  includes  all  rod- 
shaped  organisms.  Two  other  names  are  used  by  some  authors, 
namely,  Bacterium  and  Pseudomonas.  Bacterium  is  defined  by 
some  authors  as  a  non-motile  bacillus,  by  others  as  a  non-spore- 


CLASSIFICATION   OF   MICROORGANISMS 


79 


bearing  bacillus.  Sometimes  a  non-motile  organism  is  found  to 
be  physiologically,  culturally,  and  morphologically  closely  related 
to  some  motile  form,  and  it  seems  to  be  undesirable  to  separate 
these  into  different  genera.  The  differentiation  on  the  basis  of 
spore  formation  has  not  been  generally  accepted  by  bacteriologists. 
The  term  Pseudomonas  is  sometimes  used  to  indicate  a  motile 


0. 


Fig.  43. — Types  of  bacilli:  a,  6,  Non-motile  bacilli  (Bacterium);  c,  mono- 
trichous  bacillus  (Pseudomonas);  d,  lophotrichous  bacillus  (Pseudomonas); 
e,  f,  peritrichous  Bacillus. 

bacillus  having  polar  flagella,  while  the  term  Bacillus  is  limited 
to  organisms  having  flagella  over  the  entire  surface  of  the  body 
(peritrichous).  The  term  Bacillus  is  here  used  to  include  both 
Bacterium  and  Pseudomonas. 

Spirillum. — The  family  Spirillaceae  is  divided  by  Migula  into 
four  genera:  Spirosoma  is  non-motile.  Microspira  is  a  rigid, 


Fig.  44. — Types  of  spirilla:  a,  Non-motile  spirillum  (Spirosoma);  6,  mono- 
trichous  spirillum  (Microspira,  Vibrio) ;  c,  lophotrichous  spirillum  with  2  or  3 
flagella  (Microspira,  Vibrio);  d,  lophotrichous  spirillum  (Spirillum). 

short,  comma-shaped  organism  with  one,  two,  or  three  flagella. 
Vibrio  is  sometimes  used  as  a  synonym  of  microspira.  Spirillum 
is  used  to  indicate  a  long,  rigid,  spiral  bacterium  with  a  tuft  of 
flagella  at  one  or  both  ends.  As  used  in  this  text,  the  term  Spir- 
illum will  include  Spirosoma,  Microspira,  Vibrio,  and  Spirillum. 


80 


VETERINARY   BACTERIOLOGY 


Spirochaeta. — Spirochseta  is  defined  as  an  organism  with 
long,  thin,  flexible  spirals  on  which  flagella  are  demonstrated 
with  difficulty,  if  at  all.  It  is  problematic  whether  the  spirochsetae 
belong  to  the  true  bacteria  or  are  a  group  intermediate  between 
the  bacteria  and  protozoa.  Conclusive  evidence  on  this  subject 


Fig.  45. — Types  of  spirochaetae. 

is  still   lacking.     These   organisms   will   be   discussed   with   the 
protozoa. 

Chlamydobacteriaceae. — The  classification  here  given  is  that 
used  by  Jordan  in  his  "  General  Bacteriology."  Nomenclature 
of  the  forms  belonging  to  this  group  is  badly  mixed,  and  the  same 
terms  have  been  used  by  different  authors  with  reference  to  very 


Fig.  46.— A,  Leptothrix;  B,  Cladothrix;  C,  Nocardia;  D,  Actinomyces  or  Strep- 

tothrix. 


different  organisms.  Leptothrix  is  a  sheathed  organism  which 
shows  no  branching.  Cladothrix  is  one  which  shows  false 
branching.  This  false  branching  arises  through  one  of  the  inter- 
calary cells  dividing  and  pushing  the  cells  lying  above  it  to  one 
side,  and  developing  thereafter  as  a  terminal  cell.  Nocardia  in- 
cludes those  forms  in  which  there  is  true  branching,  and  in  which 


CLASSIFICATION   OF   MICROORGANISMS  81 

arthrospores  have  been  observed.  These  are  formed  usually 
by  aerial  hyphaB  which  are  thrown  up  from  the  surface  of  the 
medium  upon  which  the  organism  is  growing  and  break  up  into 
segments  which  resemble  bacilli.  The  term  Streptothrix  is  some- 
times used  as  synonymous  with  both  the  terms  Nocardia  and  Ac- 
tinomyces,  but  not  correctly,  as  the  name  Streptothrix  was,  as 
long  ago  as  1839,  used  for  a  genus  in  a  wholly  unrelated  group 
of  fungi.  Actinomyces  includes  those  types  which  show  true 
branching  and  in  which  spore  formation  has  not  been  observed. 
Probably  Nocardia  and  Actinomyces  represent  but  a  single  genus. 
In  addition  to  the  forms  here  given,  several  other  genera  have 
been  recognized  by  various  writers,  but  are  of  no  importance  to 
the  veterinarian. 

Thiothrix  includes  all  thread-like  bacteria  which  possess  no 
sheath    and   whose  cells  contain  sulphur    granules.     Conidia    or 


A 
Fig.  47.— A,  Beggiatoa  (after  Winogradsky) ;  B,  Thiothrix  (after  Ellis). 

arthrospores  are  produced  at  the  ends  of  the  threads.  This  genus 
is  of  little  importance. 

Beggiatoa. — This  includes  all  those  forms  in  which  the  cells 
are  surrounded  by  a  sheath  and  contain  sulphur  granules. 

In  addition  to  the  names  of  the  genera  given  above,  a  large 
number  of  physiologic  and  pathologic  names  have  been  given. 
For  example,  Streptococcus  pneumonia  is  frequently  referred 
to  as  the  pneumococcus.  This  is  not  in  any  sense  a  scientific 
name,  but  simply  a  convenient  term  for  common  designation. 
Other  examples  of  similar  type  are  gonococcus  for  the  specific 
cause  of  gonorrhea,  meningococcus  for  the  organism  causing 
epidemic  cerebrospinal  meningitis,  and  urobacillus  for  organisms 
causing  certain  ammoniacal  fermentations  in  urine.  Such  ex- 
amples might  be  multiplied  indefinitely. 


82  VETERINARY  BACTERIOLOGY 

CLASSIFICATION  OF  YEASTS 

Mycologists  recognize  a  number  of  different  genera  in  the  group 
of  yeasts.  It  is  probable  that  the  yeasts  do  not  constitute  a 
homogeneous  group.  The  genus  Saccharomyces  includes  such 
forms  as  the  common  bread  and  brewer's  yeast  (Saccharomyces 
cerevisice)  which  produce  spores  and  are  active  in  alcoholic  fer- 
mentation. The  name  Torula  is  sometimes  given  to  similar  yeasts 
that  are  not  spore  producing.  This  latter  name,  however,  is 
incorrectly  so  applied,  as  it  was  previously  and  is  now  used  to 
indicate  a  genus  of  molds.  The  name  Blastomyces  has  been 
commonly  accepted  to  indicate  the  yeasts  pathogenic  for  man 
and  animals.  It  is  probable  that  there  is  little  reason  for  separation 
of  Saccharomyces  and  Blastomyces  on  the  basis  of  their  morphology, 
but  such  a  separation  on  the  basis  of  pathogenesis  seems  to  be 

advisable. 

CLASSIFICATION  OF  THE  MOLDS 

Several  hundred  genera  and  many  thousands  of  species  have 
been  described.  Of  these,  a  few  genera  only  contain  species  that 
are  pathogenic  for  man  and  animals.  For  a  discussion  of  classi- 
fication the  student  is  referred  to  Chapter  XXXVIII. 


SECTION    II 
LABORATORY  METHODS  AND  TECHNIC 


CHAPTER  VI 

STERILIZATION 

STERILIZATION  is  the  process  whereby  glassware,  media,  or 
any  of  the  materials  or  apparatus  used  in  the  laboratory  are 
entirely  freed  from  living  organisms.  It  is  evident  that  in  the 
study  of  bacteria  it  is  necessary  that  we  deal  with  pure  cultures, 
that  is,  that  one  kind  of  organism  only  be  present  in  the  material 
which  we  are  studying.  It  is  quite  impossible  to  determine  from 
mixed  cultures  which  of  the  organisms  present  bring  about 
observed  changes.  Bacteria  are  present  upon  the  surface  of 
all  laboratory  apparatus,  in  the  dust,  in  soil,  upon  the  hands — 
they  are  ubiquitous,  hence  the  necessity  for  sterilization. 

Sterilization  may  be  accomplished  by  physical  or  chemical 
means.  In  practice  the  latter  is  generally  called  disinfection,  and 
is  rarely  used  in  the  laboratory.  The  term  sterilization,  there- 
fore, as  commonly  used,  indicates  the  destruction  of  micro- 
organisms by  physical  processes. 

Sterilization  by  the  Flame. — The  platinum  wire  used  in  the 
transfer  of  bacteria  in  the  laboratory  is  sterilized  by  heating  to 
a  red  or  white  heat  in  the  flame  of  the  Bunsen  burner.  Similar 
methods  are  sometimes  used  in  the  sterilization  of  other  small 
pieces  of  laboratory  apparatus,  such  as  cover-glasses  and  slides. 

Sterilization  by  Hot  Air. — Glassware  is  commonly  sterilized 
by  subjecting  it  to  a  temperature  of  150°  to  170°  in  a  hot-air 
oven  for  an  hour.  All  bacteria  will  be  destroyed  at  this  tempera- 
ture providing  the  material  to  be  sterilized  is  of  a  nature  such  that 
the  heat  can  penetrate  readily  to  all  parts.  This  method  cannot  be 

83 


84 


VETERINARY   BACTERIOLOGY 


used,  however,  in  the  sterilization  of  liquids  or  of  any  organic  mater- 
ial which  might  be  decomposed  at  such  a  temperature. 

Sterilization  by  Streaming  Steam. — It  is  found  in  practice  that 
live  steam  is  the  most  efficient  sterilizing  agent  for  many  of  the 
media  used  in  the  laboratory.  Steam  under  atmospheric  pressure 
at  sea-level  has  a  temperature  of  about  100°.  Some  type  of 
apparatus  is  used  such  that  the  live  steam  comes  in  direct  con- 
tact with  the  material  to  be  sterilized.  One  type  of  the  apparatus 
is  called  the  Arnold  steam  sterilizer  (Fig.  49).  It  consists  essen- 
tially of  a  pan  with  a  double  bottom  opening  into  the  sterilizing 


Fig.  48. — Oven  for  sterilization  by  hot  air  (Jordan). 

chamber  above.  The  water  between  the  bottoms  is  quickly 
heated  to  boiling  temperature  and  is  automatically  replaced 
from  the  supply  on  the  exterior  through  small  holes  as  rapidly 
as  it  boils  away.  A  single  exposure  to  live  steam  for  fifteen 
minutes  is  sufficient  to  kill  all  vegetative  bacteria,  but  spores 
are  not  thus  destroyed.  It  is  customary,  therefore,  to  heat  for 
fifteen  minutes  on  one  day,  keep  the  medium  for  twenty-four 
hours  at  a  temperature  suitable  for  the  germination  and  devel- 
opment of  any  spores  present,  then  heat  again  for  fifteen  minutes 
in  the  same  manner.  Those  spores  which  have  germinated  will 


'  STERILIZATION  85 

be  destroyed  by  this  second  heating.  A  third  heating,  twenty-four 
hours  later,  will  quite  certainly  destroy  all  the  bacteria  which  may 
have  been  present.  This  process  is  called  intermittent  steriliza- 
tion. It  finds  its  principal  application  in  the  sterilization  of 
materials  which  would  be  changed  or  broken  down  by  heating 
at  a  higher  temperature.  Among  such  materials  are  media  con- 
taining sugars  which  undergo  incipient  caramelization  when 
heated  too  hot. 

Sterilization  by   Steam   under   Pressure. — This   is   generally 
accomplished  in  the  autoclave  or  digester,  which  consists  essentially 


Fig.   49. — Arnold  steam  sterilizer    (Fowler). 

of  a  chamber  into  which  steam  under  pressure  can  be  introduced 
(Fig.  50).  Many  different  types  of  these  autoclaves  have  been 
put  upon  the  market.  Live  steam  under  a  given  pressure  unmixed 
with  air  has  a  constant  temperature;  therefore,  if  the  pressure 
of  the  steam  is  known,  one  can  determine  easily  the  temperature 
as  well.  It  is  necessary,  however,  that  all  air  be  first  eliminated. 
This  is  accomplished  by  allowing  the  stop-cock,  which  is  always 
present  upon  the  steam  chamber  in  the  autoclave,  to  remain  open 
until  all  the  air  has  escaped  and  the  steam  issues  in  a  constant 
stream.  This  cock  is  then  closed  and  the  pressure  caused  to 


86 


VETERINARY   BACTERIOLOGY 


rise  as  quickly  as  possible  to  15  pounds  to  the  square  inch,  or 
one  additional  atmosphere.  This  gives  a  temperature  of  about 
121°.  Material  to  be  sterilized  should  be  allowed  to  remain 
fifteen  minutes  usually.  If  large  bulks,  such  as  flasks  of  media, 
are  to  be  sterilized,  a  longer  period  must  be  allowed  in  order  that 


Fig.  50. — Autoclave  for  sterilizing  by  steam  under  pressure. 

the  media  may  be  completely  heated  through.  If  very  small 
quantities  of  material  are  bein^  sterilized,  a  shorter  period  may 
be  used.  When  properly  carried  out,  sterilization  by  this  method 
will  certainly  destroy  all  the  bacteria  present.  Its  principal 
disadvantage  is  that  certain  organic  substances  may  be  decomposed 
at  this  temperature. 


STERILIZATION 


87 


Sterilization   at   Temperatures   Lower   than   Boiling-point. — 

It  is  sometimes  necessary  to  sterilize  media,  particularly  blood- 
serum,  at  temperatures  lower  than  the  boiling-point  of  water. 
This  is  accomplished  by  placing  the  material  to  be  sterilized  in  an 
apparatus  where  it  may  be  heated  to  the  desired  temperature, 
usually  70°-80°  for  one  to  two  hours  on  each  of  five  or  more 
successive  days.  If  large  numbers  of  spores  of  certain  organisms, 
such  as  Bacillus  subtilis,  are  present,  it  is  almost  impossible  to 
sterilize  efficiently  by  this  method.  However,  if  care  is  used  in 
securing  the  blood-serum  to  prevent  the  introduction  of  such  organ- 
isms, sterilization  may  be  easily  accomplished  at  this  tempera- 
ture. 

Sterilization  by  Addition  of  Chemicals. — It  is  only  under  excep- 
tional  conditions   that   chemicals   are   used   to   sterilize   media. 


Fig.  51. — Apparatus  for  sterilization  by  filtration  (McFarland). 

It  has  been  found  that  the  addition  of  soluble  materials,  such 
as  lactose,  in  considerable  quantities  to  media  containing  pure 
cultures  of  certain  bacteria  will  destroy  the  organisms  so  that 
they  may  be  used  as  a  vaccine.  This  method  does  away  with 
the  destruction  of  any  of  the  characteristic  metabolic  products 
by  heat. 

Sterilization  by  Filtration. — Bacteria  may  be  removed  from  a 
liquid  by  passing  it  through  a  filter  with  pores  so  fine  that  the 
organism  cannot  penetrate.  Such  filters  are  made  up  in  a  great 
variety  of  shapes  and  densities.  Among  the  many  used  are  the 
Berkefeld,  the  Pasteur,  and  the  Chamberland.  These  are  made  of 
unglazed  porcelain.  In  filtration  through  these  it  is  necessary,  of 
course,  that  all  the  apparatus  used,  particularly  the  vessel  into 
which  the  filtrate  runs  and  the  filter  itself,  be  sterilized  before  use. 


88  VETERINARY   BACTERIOLOGY 

This  method  of  sterilization  is  commonly  used  for  the  removal  of 
bacteria  from  culture-media  when  it  is  desired  to  study  their 
soluble  metabolic  products,  and  in  the  removal  of  bacteria  from 
sera  which  contain  antitoxins  and  other  antibodies.  It  is  not 
commonly  used  in  the  sterilization  of  media  intended  for  the 
cultivation  of  bacteria.  Filters  of  this  character  have  been  used 
extensively  in  the  filtration  of  water  for  drinking  purposes.  When 
first  installed,  they  are  quite  efficient,  but  it  is  found  that  the  organ- 
isms rapidly  penetrate,  and  in  the  course  of  time  are  found  in  the 
filtrate.  Such  filters  must,  therefore,  be  sterilized  at  intervals  if 
they  are  to  remain  efficient. 


CHAPTER  VII 

CULTURE-MEDIA  AND  THEIR  PREPARATION 

MICROSCOPIC  examination  alone  is  quite  insufficient  to 
differentiate  species  of  bacteria.  By  the  aid  of  a  microscope 
one  cannot  readily  recognize  the  differences,  for  example,  between 
the  organisms  which  cause  typhoid  fever  and  certain  of  the  normal 
inhabitants  of  the  intestinal  tract.  It  is  necessary,  therefore, 
in  a  study  and  differentiation  of  species,  that  we  make  use  of 
different  kinds  of  culture-media  in  which  the  bacteria  may  be 
grown.  By  the  term  medium  is  meant  any  nutrient  substance  or 
mixture  upon  which  or  in  which  bacteria  will  multiply.  The 
bacteria  in  their  development  on  the  various  media  show  certain 
growth  reactions  which  are  very  useful  in  their  differentiation. 
Some  produce  acids,  others  gas,  alkalis,  and  proteolytic  and 
coagulative  enzymes. 

Use  of  Normal  Solutions  of  Acid  and  Alkali  and  Methods  of 
Expressing  Reactions. — In  the  chapter  on  Physiology  we  have 
noted  that  many  bacteria  are  extremely  sensitive  with  respect  to 
the  acidity  or  alkalinity  of  the  medium  in  which  they  are  grown. 
Some  organisms  develop  best  in  a  medium  which  is  approximately 
neutral;  some  refuse  to  develop  unless  there  is  a  slight  excess  of 
alkali  present.  It  is  necessary,  therefore,  that  some  definite  method 
of  expression  of  these  acidities  and  alkalinities  be  adopted.  For 
this  purpose  it  is  customary  to  use  normal  solutions. 

A  normal  solution  of  a  chemical  may  be  defined  as  one  in  which 
there  is  one  gram  of  replaceable  (acid)  hydrogen  or  its  equivalent 
per  liter  of  solution.  For  example,  if  we  wish  to  prepare  a  normal 
solution  of  HCl,we  must  so  dilute  the  acid  that  it  contains  one  gram 
of  hydrogen  per  liter  of  solution.  This  is  best  accomplished  in 
any  substance  that  can  be  readily  weighed  by  dissolving  the 
molecular  weight  expressed  in  grams  in  sufficient  water  to  make  a 
liter  of  solution.  If  there  is  more  than  one  atom  of  replaceable 
hydrogen  in  the  molecule,  it  is  necessary  to  divide  the  amount 
used  by  the  number  of  any  such  atoms.  For  example,  the  molecu- 


90  VETERINARY   BACTERIOLOGY 

lar  weight  of  H2SO4  is  approximately  98,  but  there  are  two  re- 
placeable acid  hydrogen  atoms.  Therefore,  half  this  molecular 
weight  in  grams,  or  49  grams,  of  the  H2S04  is  made  up  to  a  liter 
of  solution  and  contains  one  gram  of  acid  hydrogen.  The  same 
principle  is  adopted  in  the  preparation  of  a  normal  solution 
of  an  alkali;  in  this  case,  however,  it  is  necessary  to  divide  the 
molecular  weight  by  the  number  of  atoms  of  the  base  present^ 
which  will  replace  hydrogen.  For  example,  the  molecular  weight 
of  NaOH  is  40.  It  contains  one  atom  only  of  sodium,  and  a  normal 
solution,  therefore,  contains  40  grams  to  the  liter.  Dry  sodium  car- 
bonate (NajCOs)  has  a  molecular  weight  of  106.  Two  atoms  of  sod- 
ium are  present ;  therefore  it  is  necessary  to  divide  by  two,  so  that 
a  normal  solution  of  sodium  carbonate  contains  53  grams  to  the 
liter  of  solution.  It  is  evident  that  a  given  volume  of  a  normal 
solution  of  an  acid  will  neutralize  exactly  an  equal  volume  of  a 
normal  alkali. 

It  is  customary  to  use  indicators  in  the  determination  of  the 
acidity  or  alkalinity  of  a  solution.  Those  most  commonly  used 
are  litmus,  which  is  blue  for  alkaline  and  red  for  acid  solutions, 
and  phenolphthalein,  which  is  colorless  with  acid  and  red  with 
alkali.  Phenolphthalein  is  so  delicate  an  indicator  that  it  is 
sensitive  to  the  presence  of  CO2  in  solution.  It  is,  therefore, 
necessary,  whenever  this  indicator  is  used,  to  heat  the  solution 
to  boiling  temperature  in  order  to  drive  off  any  C02  which  may 
be  present.  It  is  customary  to  express  the  acidity  or  alkalinity 
of  a  solution  in  terms  of  the  amount  of  normal  acid  or  alkali  present 
per  100  c.c.  of  solution.  For  example,  if  it  is  found  that  it  requires 
10  c.c.  of  the  normal  solution  of  alkali  to  neutralize  100  c.c.  of  a 
given  solution,  we  know  that  there  is  present  in  that  solution  the 
equivalent  of  10  c.c.  of  normal  acid,  and  the  reaction  is  expressed 
as  +  10.  If  the  reaction  is  alkaline,  the  negative  sign  is  used. 

Nature  of  Nutrients  Required  by  Bacteria.— It  is  found  that 
practically  the  same  elements  are  necessary  for  the  nutrition  of 
bacteria  as  are  essential  for  higher  plants  and  animals,  but  they 
may  be  used  in  quite  different  proportions. 

It  is  particularly  important  that  the  disease-producing  bacteria 
be  cultivated  whenever  possible.  Cultivation  outside  the  body  is 
quite  necessary  to  a  satisfactory  proof  of  pathogenicity,  to  differ- 


CULTURE-MEDIA   AND   THEIR   PREPARATION  91 

entiate  species,  and  to  secure  the  organism  in  quantities  sufficient 
for  preparation  of  vaccines,  antitoxins,  etc.  A  few  standard  media 
are  commonly  used  in  the  laboratory  for  the  growth  of  bacteria,  and 
a  great  variety  of  special  types  have  been  devised  for  certain 
species  that  do  not  grow  upon  these.  It  is  impracticable  even 
to  enumerate  the  many  special  media  that  have  been  employed. 

LIQUID  MEDIA 

Bouillon  or  Beef  Broth  from  Meat. — This  is  the  commonest  of 
laboratory  media  and  serves  as  a  basis  for  the  preparation  of 
many  others. 

Place  500  gm.  chopped  lean  beef  in  a  liter  of  water  and  allow 
it  to  stand  in  a  refrigerator  over  night.  The  juice  is  then  pressed 
out  with  a  meat  press,  boiled  for  half  an  hour,  the  coagulated 
albumins  filtered  out,  the  liquid  made  up  to  a  liter  with  water,  10 
gm.  of  peptone  added,  and  heated  sufficiently  to  dissolve.  The 
reaction  is  adjusted  to  the  proper  point,  usually  4-  1,  by  titra- 
tion,  or  the  medium  is  simply  neutralized  by  addition  of  nor- 
mal XaOH,  using  phenolphthalein  paper  as  an  indicator  if  a  high 
degree  of  accuracy  is  not  required.  The  broth  is  then  autoclaved 
at  15  pounds  pressure  or  boiled  for  fifteen  minutes,  allowed  to 
cool,  and  then  filtered.  The  cooling  throws  down  a  precipitate 
of  magnesium  ammonium  phosphate,  which  may  then  be  removed. 
In  many  cases  this  is  not  objectionable  and  filtration  may  be 
carried  out  while  the  solution  is  still  hot.  The  finished  bouillon 
or  broth  is  placed  in  test-tubes  and  flasks,  and  sterilized  in  the 
autoclave  under  a  pressure  of  15  pounds  for  15  minutes. 

Bouillon  or  Broth  from  Beef  Extract. — It  is  customary,  in  much 
of  the  routine  work  of  the  laboratory,  to  substitute  for  the  pre- 
ceding a  broth  in  which  three  grams  of  a  beef  extract,  such  as 
Liebig's,  is  substituted  for  the  meat. 

Sugar-free  Broth. — There  is  generally  present  in  the  preceding 
media  a  small  amount  of  carbohydrate,  largely  dextrose.  In 
some  cases  a  sugar-free  medium  is  required.  Theobald  Smith  has 
devised  a  modification  of  the  meat  broth  for  this  purpose  which 
is  commonly  used.  Several  broth  tubes  containing  vigorous 
twenty-four-hour  cultures  of  Bacillus  coli  are  added  to  the  meat 
infusion  and  kept  at  37°  for  eighteen  hours.  In  this  time  the 


92  VETERINARY   BACTERIOLOGY 

bacteria  will  have  used  up  all  the  sugar  present.  The  broth  is 
then  prepared  as  above. 

Sugar  Broth. — Sugar-free  broth  is  generally  modified  by  the 
addition  of  carbohydrates,  such  as  dextrose,  saccharose,  and 
lactose,  making  1  per  cent,  solutions.  Such  media  must  be 
subjected  to  intermittent  sterilization  in  flowing  steam  and  not 
in  the  autoclave,  as  the  carbohydrates  readily  decompose. 

Glycerin  Broth. — Five  or  6  per  cent,  of  glycerin  added  to 
broth  makes  it  a  much  more  favorable  medium  for  many  organisms. 

Serum  Broth. — Blood-serum  secured  under  strict  aseptic  pre- 
cautions may  be  added  to  sterile  broth  in  various  proportions. 
Tubes  prepared  in  this  manner  should  be  incubated  for  a  few 
days  to  determine  whether  or  not  the  medium  is  sterile.  Steril- 
ization can  be  effected  only  by  filtration,  as  heating  would  coagulate 
the  serum. 

Dunham's  Solution. — This  is  a  solution  containing  1  per  cent, 
peptone  and  0.5  per  cent,  sodium  chlorid  in  water.  It  is  used  in 
growing  organisms  for  the  determination  of  indol. 

Beerwort. — Unhopped  beerwort  is  frequently  used  for  the 
growth  of  yeasts  and  molds. 

Milk. — Fresh  separated  milk  is  tubed  and  subjected  to  intermit- 
tent sterilization.  Commonly,  litmus  is  added  in  sufficient  quan- 
tities to  make  the  milk  a  distinct  blue. 

Synthetic  Media. — It  is  sometimes  desirable  to  prepare  a 
medium  in  which  the  exact  chemical  composition  of  every  ingre- 
dient is  known.  The  nature  of  all  the  changes  brought  about  by 
bacteria  can  be  studied  chemically  in  such  a  medium,  and  the 
food  requirements  ^  determined  by  changes  in  the  composition. 
Most  synthetic  media  contain  as  a  basis  an  aqueous  solution 
of  certain  salts,  among  them  potassium  phosphate  and  sodium 
chlorid.  Special  media  of  this  kind  are  used  extensively  in  the 
study  of  the  soil  bacteria;  it  is  only  occasionally  that  such  a 
medium  proves  serviceable  in  the  study  of  pathogenic  forms.  The 
most  commonly  used  of  the  synthetic  media  is  Uschinsky's  solution. 

Water,  distilled 1000  c.c. 

Asparagin 4  gin . ; 

Ammonium  lactate 6  gm.; 

NajHPO, 2gm.; 

NaCl 


CULTURE-MEDIA   AND   THEIR   PREPARATION  93 

LlQUEFIABLE  SOLID   MEDIA 

Nutrient  Gelatin. — This  is  prepared  by  the  addition  of  10-15 
per  cent,  of  gelatin  to  bouillon  as  prepared  above.  The  gelatin 
should  be  the  best  "  gold  label."  Care  must  be  used  in  heating 
the  solution  while  dissolving  the  gelatin  or  the  latter  will  stick 
to  the  bottom  of  the  vessel  and  burn.  It  is  best  to  use  an  asbestos 
pad,  a  double  boiler,  or  a  rice-cooker.  The  gelatin  is  itself  acid, 
so  that  it  is  necessary  to  adjust  the  reaction  after  it  has  dissolved. 
The  medium  is  then  cooled  to  60°,  and  the  white  of  an  egg  thor- 
oughly mixed  with  it.  It  is  again  heated  to  the  boiling-point 
without  stirring.  The  coagulation  of  the  egg  removes  suspended 
dust-particles  and  makes  nitration  easier.  The  nutrient  gelatin 
is  tubed  and  sterilized  in  the  autoclave  at  120°  for  ten  minutes. 
It  should  be  cooled  at  once  after  removal  from  the  sterilizer. 
Care  must  be  exercised  not  to  heat  the  medium  too  long  or  it 
may  fail  to  solidify  when  cooled. 

Other  Gelatin  Media. — Any  of  the  liquid  media  already  dis- 
cussed, with  the  exception  of  the  milk  and  serum  broth,  may  be 
made  solid  by  the  addition  of  10  to  15  per  cent,  gelatin.  Among 
the  more  commonly  used  are  dextrose,  lactose,  and  glycerin 
gelatin. 

Nutrient  Agai'. — This  is  prepared  by  the  addition  of  1.5  per 
cent,  of  shredded  or  powdered  agar-agar  to  bouillon.  Agar-agar 
is  a  carbohydrate-like  material,  probably  related  to  the  vegetable 
gums,  which  is  prepared  from  certain  of  the  seaweeds  of  the 
Pacific  and  Indian  Oceans.  The  mixture  must  be  boiled  vigorously 
for  half  an  hour  to  insure  thorough  solution  of  the  agar.  This 
medium  does  not  burn  as  readily  as  does  gelatin,  and  long- 
continued  heating  does  not  interfere  with  its  solidification  when 
cooled.  The  nutrient  agar  may  be  sterilized  in  the  autoclave 
for  fifteen  minutes  at  120°. 

Other  Agar  Media. — Agar  may  be  used  as  the  solidifying  agent 
for  any  of  the  liquid  media  described.  It  has  the  advantage 
over  gelatin  that  it  may  be  kept  at  blood  heat,  while  gelatin  under 
such  conditions  would  liquefy. 


94 


VETERINARY   BACTERIOLOGY 


NON-LIQUEFIABLE  MEDIA 

Potato. — Cylinders  are  cut  from  potatoes  by  means  of  an  apple- 
corer  or  special  potato  borer.  These  are  divided  by  a  diagonal 
longitudinal  cut  such  that  each  half  has  one  long  sloping  surface. 
It  is  well  to  soak  in  running  water  for  a  few  hours  to  prevent 
their  turning  dark  when  sterilized.  They  are  placed  with  the 
sloping  surface  up  in  test-tubes  with  a  bit  of  saturated  absorbent 
cotton  in  the  bottom,  or  in  special  potato  tubes.  The  latter 
are  tubes  constricted  a  short  distance  from  the  bottom.  The 
bulb  thus  formed  is  filled  with  water  and  the  potato  rests  on  the 
constriction  above.  This  device  enables  one  to  keep  the  potatoes 


Fig.  52. — Preparation  of  potato  tubes:  a,  Potato  cylinder  cut  diagonally;  6, 
side  view   in  tube;  c,   front  view. 

moist  for  considerable  periods.  They  are  sterilized  in  the  auto- 
clave for  fifteen  minutes  at  120°. 

Other  Vegetable  Media. — Carrots  and  other  vegetables  may 
be  prepared  in  the  same  manner  as  potato. 

Blood-serum. — Solidified  blood-serum  has  been  found  to  be 
essential  to  the  growth  in  the  laboratory  of  certain  of  the  patho- 
genic bacteria.  It  is  best  to  avoid  all  the  initial  contamination 
of  the  serum  possible,  as  it  is  difficult,  by  the  methods  used  in 
sterilization,  to  rid  the  medium  of  all  the  spore  producers  when 
they  are  present  in  considerable  numbers.  The  blood,  usually 
from  beef,  is  allowed  to  clot,  and  the  clear,  straw-colored  serum 
removed.  A  clear,  solidified  serum  may  be  prepared  by  heating 


CULTURE-MEDIA    AND   THEIR    PREPARATION  95 

the  slanted  tubes  to  76  °  for  an  hour  or  more  on  five  or  six  consecu- 
tive days.  An  opaque  medium  is  secured  by  heating  to  a  tempera- 
ture of  95°. 

Loeffler's  blood-serum  is  a  mixture  of  three  parts  of  the  serum 
with  one  part  of  neutral  1  per  cent,  dextrose  broth.  It  is  solidi- 
fied in  the  same  manner  as  the  simple  serum. 

Egg  Medium. — This  medium  was  developed  by  Dorset,  of  the 
U.  S.  Bureau  of  Animal  Industry.  It  has  come  into  common 
use  for  the  growth  of  the  Bacillus  tuberculosis  and  has  been  used  in 
recent  years  as  a  satisfactory  substitute  for  blood-serum.  Dorset's 
description  of  the  method  of  preparation  follows:  "  The  egg 
shell  is  broken  carefully,  and  the  entire  contents  dropped  into  a 
wide-mouthed  sterile  flask.  The  yolk  may  be  broken  with  a 
sterile  platinum  wire.  Gentle  shaking  of  the  flask  will  serve 
to  mix  the  white  and  yolk  of  the  egg  quite  thoroughly.  Care 
should  be  taken,  however,  not  to  shake  the  flask  so  that  a  foam  will 
be  produced,  otherwise  an  uneven  and  unsatisfactory  surface  will 
be  obtained  when  the  medium  is  hardened.  When  the  mixing 
is  complete,  the  egg  is  poured  into  tubes,  care  being  taken  to  avoid 
foaming,  and  the  tubes  containing  about  10  c.c.  of  the  medium  are 
then  inclined  in  a  blood-serum  oven  and  hardened  at  a  temperature 
of  70°  C.  This  hardening  will  usually  require  two  days,  four  or 
five  hours  each  day.  Sterilization  will  be  accomplished  at  the 
same  time.  A  higher  temperature  may  be  used  and  the.  medium 
will  be  hardened  more  quickly.  The  growth  of  tubercle  bacillus 
seems  to  be  more  vigorous  when  the  egg  is  hardened  at  70°  to 
74°  C.,  and,  in  addition,  the  prolonged  heating  probably  insures 
a  more  certain  sterilization.  The  medium  after  hardening  is 
opaque  and  yellowish  in  color,  and  usually  dry,  there  being 
practically  no  water  of  condensation  in  the  tube.  The  egg  tubes 
should  be  kept  in  an  ice-box  to  prevent  further  drying.  Just  before 
inoculation,  three  or  four  drops  of  sterile  distilled  water  should 
be  added  to  each  tube  to  supply  the  moisture  required  for  the 
satisfactory  development  of  the  tubercle  bacillus." 


CHAPTER  VIII 

BIOCHEMICAL  TESTS 

THE  physiological  characteristics  of  bacteria  are  of  considerable 
importance  hi  the  differentiation  of  species.  A  knowledge  of 
such  characteristics  is  of  assistance  in  the  isolation  and  recognition 
of  certain  species,  as  in  the  detection  of  sewage  bacteria  in  water. 

Acid  Production. — Acids  are  most  frequently  and  readily  pro- 
duced by  bacteria  in  the  presence  of  suitable  carbohydrates. 
Litmus  may  be  added  to  a  sugar  medium  for  the  detection  of  acid. 
A  quantitative  determination  of  the  acids  produced  in  a  liquid 
medium  may  be  made  by  titrating  against  decinormal  alkali. 
The  ability  of  organisms  to  produce  acid  from  various  carbohy- 
drates is  used  in  the  separation  of  the  members  of  the  intestinal 
group  of  bacteria  from  each  other.  A  record  of  the  changes  in  reac- 
tion from  time  to  time  has  been  found  valuable  in  the  differentiation 
of  the  organisms  causing  bovine  and  human  tuberculosis. 

Alkali  Production. — The  alkali  most  commonly  produced  by 
bacteria  is  free  ammonia.  It  may  be  detected  qualitatively  by 
means  of  filter  paper  dipped  in  Nessler's  solution  and  exposed  above 
the  medium.  The  ammonia  gas  turns  the  paper  brown  or  black. 
The  amounts  of  alkali  produced  may  be  determined  by  titration 
against  normal  acid. 

Gas  Production. — A  few  pathogenio»bacteria  produce  gas  from 
proteins,  but  most  gas-producing  species  require  the  presence  of 
a  carbohydrate.  The  gases  most  commonly  formed  are  carbon 
dioxid  and  hydrogen.  One  qr  more  species  of  cellulose-fermenting 
organisms  can  also  produce  methane,  and  some  of  the  denitrifiers 
free  nitrogen. 

The  ability  to  produce  gas  may  be  determined  by  inoculation 
of  the  organism  into  a  dextrose  agar  or  gelatin  tube.  Gas-bubbles 
will  appear  in  the  medium  if  the  organisms  can  ferment  dextrose. 
This  sugar  is  generally  used,  as  it  is  more  easily  fermented  than 
most  other  carbohydrates.  The  fermentation  tube  is  commonly 
used  for  the  study  of  gas  production.  The  closed  arm  is  entirely 

96 


BIOCHEMICAL  TESTS  9/ 

filled,  and  the  open  arm  partly  filled,  with  broth  containing 
the  sugar  to  be  tested.  The  gas  found  after  inoculation  col- 
lects in  the  closed  arm  and  may  be  conveniently  measured 
by  means  of  a  Frost  gasometer.  The  approximate  composition 
of  the  gas  may  be  determined  by  filling  the  open  arm  with  normal 
sodium  hydrate  and  securely  closing  the  opening  with  the  thumb, 
mixing  the  gas  with  the  alkaline  solution  by  passing  it  several 
times  from  one  arm  to  the  other,  finally  returning  it  to  the  closed 
arm  and  removing  the  thumb.  The  liquid  will  then  rise  in  the 


Fig.  53. — Fermentation  tube  and  Frost  gasometer  (Heinemann). 


closed  arm  to  replace  the  carbon  dioxid  absorbed.  The  remaining 
gas  may  be  transferred  to  the  open  arm  and  tested  by  the  flame. 
Hydrogen  is  indicated  by  a  slight  explosion.  The  relative  pro- 
portion of  carbon  dioxid  and  hydrogen  is  sometimes  of  importance 
in  the  differentiation  of  species.  More  important  still  is  the 
ability  of  a  species  to  ferment  different  kinds  of  carbohydrates. 
Some  ferment  dextrose,  but  not  lactose  or  saccharose — some  fer- 
ment two  only,  and  some  all  three. 

Reduction  Processes. — Some  bacteria,  when  living  in  the  absence 

7 


98  VETERINARY   BACTERIOLOGY 

of  free  oxygen,  can  reduce  certain  chemicals,  evidently  securing 
oxygen  for  growth  processes  by  this  means.  Litmus,  methylene- 
blue,  and  other  pigments  may  be  decolorized.  Nitrates  are 
frequently  reduced  to  nitrites.  For  this  determination  a  broth 
made  from  0.1  per  cent,  peptone  and  0.02  per  cent,  potassium 
nitrate  is  inoculated  and  incubated  for  four  days.  It  is  then 
tested  by  the  following  reagent  for  the  presence  of  nitrites: 

a.  5  N  acetic  acid  ............................  1000  c.c. 

Sulphanilic  acid  ...........................         8  gm. 

b.  5  N  acetic  acid  ............................  1000  c.c. 

Alpha-amidonaphthylene  ...................         5  gm. 

Add  2  c.c.  of  each  solution  to  the  tube  to  be  tested.  A  red  or 
rose  color  will  indicate  the  presence  of  nitrite.  A  control  in  check 
tubes  of  uninoculated  broth  should  always  be  tested  at  the  same 
time. 

In  some  cases  denitrification  goes  still  further  and  the  nitrogen 
is  liberated  in  the  free  state. 

Other  reduction  processes  have  been  described.  Among  the 
more  important  are  the  reduction  of  sulphates  to  sulphids,  and  of 
chlorates  to  chlorites. 

Indol  Production.  —  Indol  is  one  of  the  products  of  protein 
decomposition  formed  by  bacterial  action.  It  is  of  importance 
principally  because  it  may  be  demonstrated  readily  and  because 
of  the  economic  importance  of  some  of  the  bacteria  which  produce 
it.  It  is  not  formed  in  the  presence  of  sugars.  Dunham's  solu- 
tion is  inoculated  with  the  organism  to  be  tested  and  incubated 
for  several  days.  To  the  tube  are  added  a  few  drops  of  concentrated 
sulphuric  acid  and  a  cubic  centimeter  of  a  0.1  per  cent,  solution 
of  sodium  nitrite.  The  sulphuric  acid  decomposes  the  nitrite, 
freeing  nitrous  acid,  which  unites  with  the  indol  to  form  a  bright 
red  compound  known  as  nitrosoindol.  The  appearance  of  this 
characteristic  red  color  is  evidence,  therefore,  of  indol  production. 
Indol  is  an  organic  compound  of  the  empirical  formula,  C8H7N, 
and  the  structural  formula 


It  is  one  of  the  products  formed  in  intestinal  putrefaction,  and 
is  the  principal  product  which  gives  rise,  under  these  conditions, 
to  the  characteristic  "  fecal  "  odor. 


BIOCHEMICAL   TESTS  99 

Thermal  Death-point. — The  accurate  determination  of  the  exact- 
temperature  that  is  necessary  to  destroy  various  species  of  bacteria 
is  frequently  of  great  economic  importance.  Efficient  steriliza- 
tion and  pasteurization  can  be  accomplished  only  when  these  facts 
are  known.  Many  methods  have  been  suggested.  In  the  labora- 
tory the  determination  is  frequently  made  by  subjecting  freshly 
inoculated  tubes  of  broth  to  different  temperatures  in  a  water-bath 
for  ten  minutes  each.  For  reliable  results  more  accurate  methods 
are  needed.  One  of  the  commonest  and  best  is  the  use  of  the 
Sternberg  bulb.  This  is  blown  of  thin  glass.  A  definite  amount  of 
culture  is  introduced  and  the  neck  sealed  in  the  flame.  The  bulbs 
are  completely  immersed  in  a  water-bath  and  suspended  by  wires 
or  by  some  other  method,  so  that  they  do  not  come  in  contact 
with  the  walls  of  the  bath,  and  heated.  A  number  of  bulbs  are 
prepared  and  one  heated  five  minutes,  another  ten  minutes,  at  50°. 
The  temperature  is  raised  two  degrees  and  two  more  bulbs  are  ex- 
posed. For  sporeless  bacteria  the  test  should  be  made  to  70°, 
and  still  higher  for  those  that  produce  spores.  The  bulbs  are  cooled 
quickly  after  their  exposure,  and  their  contents  mixed  with  agar  in 
a  Petri  dish,  or  added  to  a  tube  of  other  suitable  medium.  This 
is  then  incubated  for  several  days.  The  minimum  temperature 
required  to  destroy  the  bacteria  can  readily  be  determined  by  a 
comparison  of  the  tubes. 

Efficiency  of  Disinfectants. — The  efficiency  of  disinfectants 
is  determined  by  testing  their  action  on  pure  cultures  of  bacteria. 
Koch's  method,  which  has  been  commonly  used,  consists  in  drying 
the  organism  on  silk  threads,  immersing  them  for  varying  lengths 
of  time  in  the  disinfectant  to  be  tested,  washing  in  sterile  water, 
and  placing  them  upon  the  surface  of  agar. 

Hill's  method  is  a  modification  of  that  of  Koch,  and  is  rather 
more  accurate  and  relatively  simple.  Sterilized  glass  rods  are 
coated  at  the  tip  with  the  bacteria  to  be  tested  by  dipping  them 
into  a  broth  culture  to  a  depth  of  an  inch.  These  are  placed  in 
test-tubes  and  carefully  dried  in  a  thermostat.  They  may  then  be 
immersed  to  a  somewhat  greater  depth  in  the  disinfectant  to  be 
tested  for  definite  periods  of  time,  rinsed  carefully  in  sterile  water, 
and  placed  in  tubes  containing  broth. 


CHAPTER  IX 

MICROSCOPIC  EXAMINATION  AND  STAINING  METHODS 

OBJECTIVES  having  a  higher  power  than  those  commonly  used 
in  other  work  are  required  for  the  examination  of  bacteria.  A 
^inch  or  1.8-2  mm.  oil-immersion  objective  is  most  commonly 
used.  This  lens  differs  from  the  low-power  dry  lenses  in  that  it 
requires  a  layer  of  cedar  oil  between  it  and  the  object  to  be  exam- 
ined. This  oil  is  used  upon  the  lens  for  the  following  reasons.  In 
general,  the  higher  the  power  of  the  objective,  the  smaller  the 


Fig.  54. — Diagram  showing  the  function  of  an  oil-immersion  objective  (adapted 

from  Gage). 

opening  through  which  light  may  come  to  the  eye.  It  is  necessary, 
therefore,  that  all  the  light  possible  shall  enter  the  lens  in  order 
that  a  well-illuminated  field  may  result.  The  accompanying  ex- 
aggerated diagrammatic  representation  of  the  objective  and  the 
stage  of  the  microscope,  may  be  helpful  in  understanding  the  use 
of  the  oil. 

Let  C  represent  the  microscopic  slide,  H  the  drop  of  oil  having 
the  same  refractive  index  as  glass,  and  L  the  tip  of  the  objective 
100 


MICROSCOPIC   EXAMINATION  '  AND    STAINING;  ^l^frliODS.      101 

with  the  opening  F'F.  The  rays  of  light  are  focused  upon  the  ob- 
ject to  be  examined  by  the  mirror  or  Abbe  condenser.  Those 
rays  of  light,  such  as  BN,  that  strike  the  glass  perpendicularly 
pass  through  and  enter  the  lens  without  any  deflection.  A  ray  of 
light,  such  as  AB,  striking  the  glass  at  a  considerable  angle,  is 
refracted  upward  and  toward  the  normal  or  in  the  direction  of  BD. 
Upon  entering  the  air  it  would  again  be  refracted  and  leave  the 
glass  in  a  direction  parallel  to  the  original  ray,  or  DE,  and  would 
not  enter  the  lens.  If,  on  the  other  hand,  a  drop  of  oil  having  the 
same  refractive  index  as  the  glass  intervenes,  there  will  be 
no  refraction  at  D,  but  the  ray  will  pass  through  to  the  opening 
of  the  lens.  This  is  represented  by  the  ray  A'BD'F'.  The  use 
of  the  oil,  therefore,  results  in  a  more  brilliantly  illuminated  field 
and  a  clearer  definition  of  the  objects  to  be  examined. 

Measuring  Bacteria. — Bacteria  may  be  measured  under  the 
microscope  in  one  of  several  ways.  A  micrometer  scale  ruled  on 
glass  may  be  inserted  in  the  ocular,  and  the  distance  between  the 
lines  determined  by  examination  of  a  micrometer  scale  ruled  upon 
the  slide  or  cover-glass  examined  under  the  microscope.  When 
the  calibration  has  been  effected,  the  ocular  micrometer  may  be 
used  to  measure  the  bacteria  directly.  The  unit  of  microscopic 
measure  is  the  micron,  the  one-thousandth  part  of  a  millimeter. 

Examination  of  Living  Bacteria. — Hanging  Drops. — The  deter- 
mination of  the  motility  of  bacteria  can  best  be  accomplished  by 
the  examination  of  the  living  cells  under  the  microscope.  A  hang- 
ing-drop preparation  is  commonly  used  for  the  purpose.  A  loop- 
ful  of  broth  culture  of  the  organism  to  be  tested  is  placed  upon  the 
center  of  a  carefully  cleansed  and  flamed  cover-glass.  Growth 
from  an  agar  or  other  culture  may  be  used  by  substituting  a 
drop  of  physiological  salt  solution  or  sterile  bouillon  and  introduc- 
ing a  minute  quantity  of  the  growth  on  a  platinum  needle.  This 
drop  is  then  carefully  inverted  over  the  cavity  in  a  hollow  ground 
slide,  and  sealed  with  a  little  vaselin.  It  may  be  examined  with 
a  high-power  dry  lens  or  with  the  oil-immersion  objective.  The 
drop  may  most  easily  be  brought  into  focus  at  its  margin.  The 
light  must  be  carefully  regulated  by  means  of  the  mirror  and  the 
iris  diaphragm  of  the  Abbe  condenser  to  make  the  bacteria  most 
clearly  visible. 


102  VETERINARY   BACTERIOLOGY 

Quite  as  effective  an  observation  may  be  made  in  many  cases 
by  placing  a  drop  of  the  culture  upon  a  glass  slide  and  dropping 
a  cover-glass  upon  it,  using  care  to  include  a  few  air-bubbles. 
A  film  of  liquid  sufficiently  thick  for  the  free  movement  of  the  bac- 
teria will  remain  between  the  two  glasses.  The  edges  of  the  air- 
bubbles  furnish  a  convenient  object  upon  which  to  focus. 

STAINING  METHODS 

Bacteria  as  well  as  the  pathogenic  protozoa  are  generally  so 
transparent  when  examined  in  a  living  condition  that  the  details 
of  their  morphology  can  be  made  out  only  with  difficulty.  It  is 
customary  to  stain  these  organisms  with  various  anilin  dyes 
which  render  them  distinctly  visible. 

The  stains  used  in  biological  work  are,  for  the  most  part,  known 
as  anilin  dyes,  because  they  are  derivatives  of  anilin,  C6H5NH2. 
They  are  grouped  as  acid  or  basic,  depending  on  whether  the  acid 
radical  or  the  base  possesses  the  tinctorial  powers.  Fuchsin, 
for  example,  is  a  basic  stain,  while  ammonium  picrate  is  an  acid 
stain.  The  basic  stains  are  the  more  useful  in  the  study  of  bacteria; 
the  acid  stains  are  sometimes  used  as  counterstains,  particularly 
for  tissues  in  which  the  organisms  may  be  embedded.  The 
anilin  dyes  are  of  all  the  colors  of  the  rainbow.  The  most  com- 
monly used  are  gentian-violet,  methylene-blue,  thionin  blue, 
fuchsin,  and  Bismarck-brown. 

Mordants. — Anything  which  will  cause  a  stain  to  penetrate  an 
organism  better  or  which  causes  it  to  set  is  termed  a  mordant. 
For  example,  carbolic  acid  or  anilin  added  to  certain  stains  makes 
them  more  intense.  A  solution  of  iodin  in  potassium  iodid,  a 
mixture  of  tannic  acid  and  iron  sulphate,  and  many  other  solu- 
tions are  used  under  various  conditions  as  mordants. 

Formulas  of  Some  of  the  Commonly  Used  Stains. — There  are  a 
few  stains  which  find  constant  use  in  the  laboratory.  The  formulas 
of  these  will  be  given.  There  are,  in  addition',  a  great  many  others 
which  have  special  applications. 

Loffler's  methylene-blue: 

Saturated  alcoholic  solution  of  methylone-blue 15  c.c. 

Solution  of  potassium  hydrate  (1  :  1000) 50  c.c. 


MICROSCOPIC   EXAMINATION    AND    STAINING    METHODS      103 

Aqueous  solution  of  gentian-violet: 

Saturated  alcoholic  solution  of  gentian-violet 2.5  c.c. 

Distilled  water 47.5  c.c. 

Anilin  gentian-violet  (Ehrlich's)  : 

Saturated  alcoholic  solution  of  gentian-violet 6  c.c. 

Absolute  alcohol 5  c.c. 

Anilin  water 50  c.c. 

Anilin  water  is  prepared  by  adding  2  c.c.  of  anilin  to  98  c.c.  of 
distilled  water  and  shaking  vigorously  for  several  minutes.  It 
should  then  be  filtered  until  clear. 

Carbol  or  phenol  fuchsin  (Ziehl's) : 

Saturated  alcoholic  solution  of  fuchsin 5c.c. 

Solution  of  phenol,  0.5  per  cent 45  c.c. 

Bismarck-brown:  This  is  commonly  used  as  a  saturated  aqueous 
solution. 

Gabbett's  methylene-blue: 

Methylene-blue,  dry 2  gm. 

Sulphuric  acid 25  c.c. 

Distilled  water 75  c.c. 

Preparation  of  a  Stained  Mount. — A  drop  of  water  about  the 
size  of  a  pinhead  is  placed  upon  a  clean  cover-glass.  With  a 
sterile  platinum  needle  remove  a  small  portion  of  the  material  to 
be  examined  and  mix  thoroughly  in  the  drop  of  water.  When  the 
bacteria  are  in  bouillon  or  other  liquid  media,  the  drop  of  water 
is  unnecessary.  This  is  then  spread  in  a  thin  film  over  the  surface 
of  the  glass  and  dried.  The  film  is  next  fixed  by  passing  the 
cover-glass,  film  up,  through  the  flame  of  the  Bunsen  burner  three 
times.  The  stain  is  placed  upon  the  glass  and  allowed  to  act  for 
a  few  seconds  to  ten  minutes,  depending  upon  the  organism  and 
the  stain  used.  This  is  then  washed  in  water  until  no  more  stain 
comes  off.  It  is  dried  between  filter-paper  and  placed  film  down 
upon  a  drop  of  water  on  a  slide  and  examined  under  the  microscope. 
If  satisfactory,  it  may  be  floated  off  with  water,  dried,  and  placed 
film  down  on  a  drop  of  Canada  balsam  on  the  slide. 

In  many  laboratories  the  use  of  the  cover-glass  is  largely 
dispensed  with,  and  certainly  routine  examinations  of  many  kinds 
can  be  more  conveniently  made  by  means  of  films  prepared 


104  VETERINARY   BACTERIOLOGY 

directly  upon  the  glass  microscopic  slides.  The  procedure  is 
practically  identical  with  that  detailed  above  for  cover-glass 
preparations  except  that  the  immersion  oil  may  be  placed  directly 
upon  the  stained  film  and  no  cover-glass  used. 

Spore  Stain. — Bacterial  spores  stain  with  difficulty,  but  once 
stained  do  not  yield  up  the  stain  readily.  Either  one  of  the 
following  methods  will  be  found  to  give  good  results : 

Hansen  Method. — 1.  Prepare  a  film,  fix,  and  stain  with  steam- 
ing hot  carbol-fuchsin  for  five  minutes. 

2.  Decolorize  with  5  per  cent,  acetic  acid  until  the  film  is  a  light 
pink,  and  wash  in  water. 

3.  Stain  three  minutes  with  Loffler's  methylene-blue. 

4.  Examine. 

M oiler's  Method. — 1.  Prepare  films  and  fix  in  chloroform  for 
two  minutes. 

2.  Dry  in  air. 

3.  Cover  with  5  per  cent,  solution  of  chromic  acid  for  two 
minutes. 

4.  Wash  in  water. 

5.  Stain  with  hot  carbol-fuchsin  five  minutes. 

6.  Decolorize  with  1  per  cent,  sulphuric  acid  twenty-five  to 
thirty  seconds. 

7.  Wash  and  counterstain  with  methylene-blue  ten  to  fifteen 
seconds. 

8.  Examine. 

By  either  method  the  spores  appear  red  and  the  cell  body  blue. 

Stain  for  Acid-fast  (Acid-proof)  Organisms. — Certain  bacteria 
are  stained  with  difficulty,  but  when  once  stained,  they  resist 
decolorization  with  acids.  The  most  important  of  these  organisms 
is  Bacillus  tuberculosis. 

Acid  Alcohol  Method. — 1.  Prepare  film,  fix  in  flame,  and  stain 
with  hot  carbol-fuchsin  for  two  minutes. 

2.  Wash  in  2  per  cent,  hydrochloric  acid  in  95  per  cent,  alcohol 
until  there  is  no  color  visible  in  the  thinner  portions  of  the  film. 

3.  Wash  in  water  and  stain  with  methylene-blue  for  contrast. 

4.  Wash  and  examine. 

GabbetCs  Method. — 1.  Prepare  film  and  stain  as  above  with  car- 
bol-fuchsin. 


MICROSCOPIC   EXAMINATION   AND   STAINING   METHODS      105 

2.  Wash  in  water. 

3.  Stain  with  Gabbett's  methylene-blue  for  one-half   to  one 
minute. 

4.  Wash  and  examine. 

The  acid-fast  organisms  will  be  red  in  a  blue  field. 

Flagella  Stain. — The  flagella  of  bacteria  are  not  visible  in 
ordinary  stained  mounts,  and  can  be  demonstrated  only  by  a 
special  technic.  Young,  twelve-  to  eighteen-hour  cultures  of 
bacteria  should  be  used  for  their  demonstration.  A  tube  con- 
taining a  few  cubic  centimeters  (5)  is  inoculated  with  sufficient 
quantity  of  the  growth  carefully  removed  from  the  agar  surface 
to  produce  a  slight  turbidity.  Incubate  for  an  hour  in  the  ther- 
mostat. Drop  two  or  three  drops  without  mixing  or  spreading 
on  a  clean  cover-slip.  Dry  and  then  fix  in  the  flame.  Many 
methods  of  staining  flagella  have  been  suggested;  the  two  follow- 
ing are  probably  the  best: 

Van  Ermengem's  Method. — 1.  Place  the  film  for  one  hour  in  the 
following  solution: 

Osmic  acid,  2  per  cent 1  part 

Tannin,  10-25  per  cent,  solution 2  parts 

2.  Wash  in  water,  then  absolute  alcohol,  then  place  in  the 
following  solution  for  a  few  seconds  only: 

Silver  nitrate,  0.05  per  cent,  in  distilled  water. 

3.  Wash  in  the  following  solution  for  a  few  seconds: 

Gallic  acid 5  gm. 

Tannin 3  gm. 

Fused  potassium  acetate 10  gm. 

Distilled  water 350  c.c. 

4.  Wash  in  silver  nitrate  solution  until  film  turns  black. 

5.  Wash  in  water  and  examine. 

Loffler's  Method. — 1.  Prepare  film,  fix,  and  apply  the  following 
mordant,  heating  for  five  minutes  over  a  water-bath: 

Tannic  acid  (25  per  cent,  aqueous  solution) 10  parts 

Saturated  solution  ferrous  sulphate 5  parts 

Fuchsin  (saturated  alcoholic  solution) 1  part 


106  VETERINARY   BACTERIOLOGY 

2.  Wash  and  blot  with  filter-paper. 

3.  Stain  with  hot  anilin-gentian-violet  or  carbol-fuchsin  over 
a  water-bath  for  five  minutes. 

4.  Wash  and  examine. 

Gram's  Staining  Method. — This  method  was  first  used  to 
demonstrate  bacteria  in  tissues,  the  bacteria  retaining  and  the  tis- 
sues losing  the  stain.  It  was  later  found  that  not  all  bacteria 
could  be  stained  by  this  method,  and  it  has  in  consequence  come 
into  general  use  for  separating  bacteria  into  two  groups,  termed 
respectively  gram-positive  and  gram-negative,  the  former  retain- 
ing the  stain  and  the  latter  losing  it. 

1.  Prepare  film,  dry,  and  fix. 

2.  Stain  one  and  one-half  minutes  in  anilin-gentian-violet. 

3.  Treat  with  Gram's  iodin  solution  one  and  one-half  minutes. 

lodin 1  gm. 

Potassium  iodid '. 2  gm. 

Water 300  c.c. 

4.  Decolorize  with  95  per  cent,  alcohol  for  five  minutes. 

5.  Wash,  dry,  and  mount. 

Blood  and  Protozoan  Stains. — Many  special  stains  have  been 
devised  for  demonstrating  the  blood  elements  and  protozoa  in 
the  blood  and  in  tissues.  The  chief  of  these  are  the  Romanowsky 
and  Giemsa,  each  with  numerous  modifications.  These  may  most 
profitably  be  purchased  ready  for  use  from  a  reliable  dealer. 
The  methods  of  use  will  be  discussed  in  connection  with  specific 
microorganisms . 


CHAPTER  X 

METHODS  OF  SECURING   PURE  CULTURES  OF  BACTERIA 

BACTERIA  must  be  studied  in  pure  culture  if  one  is  to  deter- 
mine with  certainty  their  cultural,  physiological,  or  pathogenic 
characters.  One  of  the  first  efforts  made  in  the  study  of  a  disease 
or  any  other  process  brought  about  by  bacteria  is  to  separate  its 
causal  organism  from  all  others.  Many  methods  have  been  devised 
for  this  purpose,  not  any  one  of  them  applicable  to  every  case. 

Dilution  Method. — This  method  of  securing  pure  cultures  is 
of  historic  interest  only.  In  the  beginnings  of  the  cultivation  of 
microorganisms  the  culture-media  commonly  used  were  liquids, 
such  as  infusions  from  meat  and  vegetables,  and  beerwort.  This 
method  was  used  most  commonly  in  securing  pure  cultures  of 
yeasts.  A  long  series  of  flasks  was  prepared  with  sterile  media. 
The  impure  culture  or  mixture  of  organisms  was  mixed  thoroughly 
with  the  contents  of  the  first  flask,  and  a  definite  amount  transferred 
from  this  to  another  flask,  from  this  to  each  of  several  others,  from 
each  of  these  into  another  group,  and  so  on.  The  last  dilution 
would,  in  general,  remain  sterile,  but  among  some  of  the  dilutions 
would  be  a  group  in  which  some  flasks  would  show  growth  and 
others  of  the  same  dilution  would  not.  The  inference  was  that 
such  a  flask  had  been  planted  with  but  a  single  organism,  and  the 
flask  contents,  therefore,  constituted  a  pure  culture.  This  method 
is  cumbersome,  uncertain,  and  is  rarely  used. 

Isolation  by  Smearing. — If  a  loopful  of  a  mixed  culture  of 
microorganisms  be  drawn  across  the  surface  of  a  solid  medium  in 
parallel  streaks,  the  first  portion  will  generally  show  a  solid  line 
of  mixed  growth,  but  farther  along  the  growth  is  discontinuous. 
Many  of  the  isolated  colonies  here  will  be  found  upon  examination 
to  consist  of  pure  cultures.  This  method  is  used  for  the  isolation 
of  bacteria  from  the  mouth  and  throat  in  some  cases. 

Direct  Isolation. — Barber  has  devised  a  capillary  pipette  method 
whereby  it  is  possible  to  pick  up  a  single  bacterial  cell  and  transfer 
it  to  a  nutrient  medium  without  any  other  organisms  being  carried 

107 


108  VETERINARY   BACTERIOLOGY 

over.  This  method  has  been  found  useful  in  the  study  of  develop- 
mental and  evolutionary  problems,  but  is  not  practicable  for 
routine  laboratory  isolations. 

Isolation  by  Plating. — The  development  by  Koch  of  the  lique- 
fiable  media  furnished  a  ready  means  for  the  isolation  in  pure 


Fig.  55. — Isolation  by  successive  streak  cultures  on  an  agar  or  gelatin 
plate:  A,  First  streak  solidly  grown;  B,  second  streak,  discontinuous;  C, 
third  streak,  having  many  isolated  colonies. 

culture  of  most  species  of  bacteria.  Nutrient  agar  or  gelatin 
or  one  of  their  modifications  may  be  used.  The  medium  is  lique- 
fied by  heat,  then  cooled  in  a  water-bath  to  about  43°.  The 
mixed  culture  of  organisms  from  which  it  is  desired  to  isolate 
pure  cultures  is  inoculated  into  one  of  the  tubes.  From  this 


Fig.  56.— Petri  dish  (McFarland). 

transfers  are  made  by  means  of  a  sterile  platinum  loop  to  a  second 
tube;  this  is  thoroughly  mixed  and  transfers  made  to  a  third  tube, 
and  from  this  even  to  a  fourth.  Each  of  these  tubes  of  media 
is  then  poured  into  a  sterile,  flat,  glass,  covered  dish  called  a  Petri 
dish.  These  Petri  dishes  or  "  plates  "  are  allowed  to  stand  until 
the  medium  has  solidified;  they  are  then  incubated  and  examined 


METHODS   OF   SECURING    PURE    CULTURES   OF   BACTERIA      109 

from  time  to  time.  The  organisms  are  separated  from  each 
other  by  this  process  of  dilution,  and  are  held  fast  by  the  solidifica- 
tion of  the  medium.  In  most  cases  the  conditions  are  favorable 
for  growth,  and  development  begins.  Within  a  few  days  sufficient 
multiplication  takes  place,  so  that  the  mass  of  organisms  that  has 
developed  from  the  single  isolated  individuals  has  reached  a  size 
that  can  be  easily  seen  with  the  unaided  eye.  Such  a  mass  of  organ- 
isms is  termed  a  colony.  Transfers  from  such  colonies  will  show 
only  a  single  kind  of  organism  present,  and  by  making  isolations 
from  each  type  of  colony,  pure  cultures  may  be  secured  of  each 
species  present. 

Isolation  by  the  Use  of  Heat. — When  it  is  desired  to  isolate  a 
spore-producing  organism  from  non-sporulating  forms,  the  culture 
may  be  heated  to  80°  for  fifteen  minutes.  This  will  not  destroy 
the  spores,  but  will  eliminate  all  other  cells.  If  one  species  of 
spore-forming  organism  only  is  present,  this  results  in  a  pure  cul- 
ture at  once ;  if  more  than  one  species,  plating  becomes  necessary. 

Isolation  by  the  Use  of  Differential  Antiseptics  or  Disinfect- 
ants.— Not  all  species  of  bacteria  are  affected  alike  by  a  given 
antiseptic  or  disinfectant,  and  it  is  sometimes  possible  to  add  a 
substance  that  will  prevent  the  growth  or  kill  one  form  without 
interfering  seriously  with  the  growth  of  others.  A  small  amount 
of  phenol  added  to  bouillon  will  inhibit  the  growth  of  most  bacteria, 
with  the  exception  of  certain  members  of  the  intestinal  group. 
A  still  better  example  of  such  substance  is  antiformin,  which, 
when  mixed  with  sputum  or  other  materials  containing  tubercle 
bacilli,  destroys  all  other  organisms  than  these,  and  enables  one  to 
secure  a  pure  culture  at  once.  This  will  be  discussed  in  greater 
detail  under  the  heading  of  Tuberculosis. 

Isolation  by  Animal  Inoculation. — Some  species  of  pathogenic 
bacteria  develop  very  slowly  upon  artificial  media,  or  require 
a  special  medium  for  their  growth.  When  these  occur  mixed 
with  other  organisms,  it  is  sometimes  difficult  to  secure  them  in 
pure'  culture.  This  difficulty  may  in  some  cases  be  overcome  by 
animal  inoculation.  The  injection  of  the  organism  into  a  suitable 
susceptible  animal  results  in  thedestruction-bythe  body  of  the  other 
bacteria  injected  at  the  same  time,  and  the  characteristic  organism 
may  later  be  isolated  in  pure  cultures  from  the  lesions  of  the  disease. 


CHAPTER  XI 


STUDY  OF  BACTERIAL  CULTURES 

SPECIES  of  bacteria  are  frequently  separable  from  each  other 
on  the  basis  of  differences  in  cultural  characters  alone.  It  is, 
therefore,  important  that  careful  descriptions  should  be  kept  of 
the  cultural  characteristics  of  each  of  the  species.  For  assistance 
in  such  descriptions  the  Society  of  American  Bacteriologists  has 
adopted  a  standard  descriptive  chart  from  which  the  following  are 

adapted : 

CULTURAL  CHARACTERS 

Agar  Stroke. — This  is  prepared  by  drawing  an  inoculated  needle 
from  the  base  to  the  top  of  the  slanted  surface  of  an  agar  tube 
that  has  solidified  in  the  sloping  position.  In  this  culture  are  to 


Fig.  57. — Types  of  growth  on  agar  slants. 

be  noted  the  abundance,  form,  elevation,  luster,  surface,  and  optical 
characters  of  the  growth,  its  pigment  production,  odor,  consistency, 
and  any  changes  that  have  occurred  in  the  medium. 

Potato. — The  potato  is  inoculated  and  the  growth  character- 
istics studied  in  the  same  manner  as  the  agar  stroke, 
no 


STUDY   OF   BACTERIAL   CULTURES 


111 


Blood-serum. — This  is  inoculated  in  the  same  manner  as  the 
agar  slope,  and  the  same  characteristics  are  to  be  noted,  with  the 
addition  of  liquefaction  or  digestion  of  the  medium. 

Gelatin  Stab. — This  is  prepared  by  running  an  inoculated  plat- 
inum needle  in  a  straight  line  from  the  surface  of  an  erect  tube 
of  nutrient  gelatin  nearly  to  the  bottom,  and  withdrawing  it  with- 
out cutting  the  medium  by  any  lateral  motion  of  the  needle. 


Fig.  58. — Potato  slant  culture  (Page,  Frothingham  and  Paige,  in 
of  Medical  Research"). 


Journal 


The  characters  to  be  noted  are  abundance  and  uniformity  of 
growth  along  the  line  of  the  stab,  the  form  of  growth,  liquefaction, 
and  other  changes  in  the  medium. 

Nutrient  Broth. — This  is  inoculated  by  shaking  an  infected 
platinum  needle  in  the  medium.  The  characters  to  be  noted  are 
abundance  and  character  of  surface  growth  and  character  of 
sediment. 

Milk. — Milk  is  inoculated  in  the  same  manner  as  the  nutrient 


112 


VETERINARY    BACTERIOLOGY 


broth.  The  characters  to  be  noted  are  presence  or  absence  of 
coagulation,  type  of  curd  produced,  whether  or  not  whey  is  extruded, 
peptonization  or  digestion  of  the  casein,  acid  production,  con- 
sistency, and  changes  in  color  of  the  medium. 

Litmus  Milk. — In  addition  to  the  preceding,   acid  or  alkali 
production  and  reduction  of  the  litmus  are  to  be  noted. 


Fig.  59. — Types  of  growth  in  stab  cultures:  A,  Non-liquefying:  1,  Filiform 
(B.  coli);  2,  beaded  (Str.  pyogenes);  3,  echinate  (Bact.  acidi  lactici);  4,  villous 
(Bact.  murisepticum) ;  5,  arborescent  (B.  mycoides).  B,  Liquefying:  6, 
Crateriform  (B.  vulgare,  twenty-four  hours) ;  7,  napiform  (B.  subtilis,  forty- 
eight  hours);  8,  infund^mliform  (B.  prodigiosus) ;  9,  saccate  (Msp.  Finkleri); 
10,  stratiform  (Ps.  fluorescens)  (Frost). 


Gelatin  Plate  Colonies. — Two  or  three  tubes  of  gelatin  are 
melted,  cooled  to  40°,  and  one  inoculated  with  a  small  amount  of 
the  organism  to  be  studied.  The  tube  is  rolled  until  the  bacteria 
are  thoroughly  distributed,  and  with  a  platinum  loop  a  transfer 
is  made  to  a  second  tube,  and  from  this  to  a  third.  The  con- 
tents of  each  tube  are  then  poured  into  a  Petri  dish  and  allowed 


STUDY    OF   BACTERIAL   CULTURES 


113 


to  solidify.     The  plate  showing  a  small  number  of  colonies  devel- 
oping is  the  one  chosen  for  examination.     The  characters  to  be 


Fig.  60. — Portion  of  an  agar  plate  culture  showing  a  mold  colony  and  five 

bacterial  colonies. 


noted  are  rapidity  of  growth,  form,  elevation,  and  edge  of  the  colony 
and  type  of  liquefaction  if  it  occurs. 


Fig.  61. — Ameboid  colony  on  an  agar  plate  (Lewton-Brain  and  Deerr). 


Colonies  on  Agar  Plates. — Plates  containing  nutrient  agar  are 
prepared  in  the  same  manner  as  the  gelatin  plates  described.     The 

8 


114 


VETERINARY   BACTERIOLOGY 


Fig.  62. — Spreading  colony  on  an  agar  plate  (Lewton-Brain  and  Deerr). 


Fig.  63. — Colonies  in  an  agar  plate  culture  (Lewton-Brain  and  Deerr). 


characters  to  be  noted  are  rapidity  of  growth,  form,  surface  ele- 
vation, edge,  and  internal  structure  of  the  colony. 


STUDY  OF  BACTERIAL  CULTURES  115 

PHYSIOLOGICAL  CHARACTERS 

It  is  customary  to  determine  gas  and  acid  production  in  car- 
bohydrate media,  development  of  ammonia,  reduction  of  nitrates 
to  nitrites,  indol  production,  temperature  relations,  including 
optimum  growth  temperature  and  thermal  death-point,  resistance 
to  desiccation  and  disinfectants,  and  pathogenic  characters.  The 
methods  of  study  of  these  characters  have  already  been  discussed. 


SECTION    III 

BACTERIA    AND  THE    RESISTANCE  OF  THE  ANIMAL 
BODY  TO  DISEASE 


CHAPTER  XII 

BACTERIA   AND  DISEASE;    GENERAL    CONSIDERATIONS 

Koch's  Rules. — The  proof  of  the  germ  theory  of  disease  may  be 
dated  from  1876,  when  Koch  succeeded  in  demonstrating  the  rela- 
tionship of  Bacillus  anthracis  to  the  disease  anthrax.  He  later 
formulated  the  rules  (which  are  known  as  Koch's  rules)  for  the 
determination  of  the  specific  relationships  of  an  organism  to  a 
disease.  They  may  be  stated  as  follows: 

1.  The  suspected  organism  must  be  found  in  every  case  of  the 
disease  under  consideration. 

2.  The  organism  must  be  isolated  and  grown  in  pure  culture. 

3.  Inoculation  of  the  organism  into  suitable  animals  should 
reproduce  the  disease. 

4.  The  organism  must  again  be  isolated  from  such  animals. 
Unfortunately,  the  proof  of  the  cause  is  still  lacking  in  a  good 
many  diseases,  and  is  unsatisfactory  in  others.     There  are  several 
reasons  for  this: 

(a)  The  organisms  in  some  cases  have  been  shown  to  be  ultra- 
microscopic  and  capable  of  passing  through  a  porcelain  filter,  as, 
for  example,  those  which  cause  rinderpest  and  hog-cholera. 

(6)  Some  organisms,  although  evidently  not  ultramicroscopic, 
have  never  been  satisfactorily  demonstrated  under  the  microscope, 
possibly  from  lack  of  proper  staining  methods. 

(c)  Some  organisms  are  specific  for  man  and  do  not  repro- 
duce disease  of  the  same  type  when  inoculated  into  animals. 
With  some  of  these,  accidental  or  intentional  inoculation  into 
man  has  supplied  the  needed  evidence. 

116 


BACTERIA    AND    DISEASE;    GENERAL    CONSIDERATIONS        117 

(d)  The  organism  may  be  demonstrated  microscopically,  but 
will  not  grow  upon  the  culture-media  of  the  laboratory.  Such 
are  some  of  the  protozoa.  With  a  few  of  these  the  proof  has  been 
perfected  by  the  study  of  the  growth  of  the  organism  in  an  inter- 
mediate host,  for  example,  the  malarial  parasite  in  the  mosquito. 

Evidence  of  the  relationship  of  an  organism  to  a  disease  may 
frequently  be  secured  by  using  the  agglutination,  precipitation, 
or  some  of  the  other  tests  discussed  in  the  following  chapters. 
Improvements  in  staining  technic  continually  reveal  new  or- 
ganisms. 

Animal  Inoculation. — Experimental  inoculation  and  injections 
are  made  in  the  bacteriological  laboratory  for  a  number  of  reasons: 

1.  In  determining  the  causal  relationships  of  a  specific  organism 
to  a  disease  in  accordance  with  Koch's  rules. 

2.  In  the  diagnosis  of  certain  diseases.     For  example,  one  of 
the  most  satisfactory  methods  of  diagnosing  glanders  is  to  inject 
some  of  the  nasal;  discharge  from  a  suspected  animal  into  a  male 
guinea-pig  and  note  the  development  of  acute  orchitis  and  subse- 
quent general  body  reaction. 

3.  In  the  isolation  of  certain  pathogenic  bacteria.     For  example, 
if  it  is  desired  to  isolate  the  organism  causing  tuberculosis  from 
sputum  or  from  milk,  it  may  most  readily  be  accomplished  by 
inoculating  the  infected  material  into  a  suitable  animal.     All  the 
non-pathogenic    organisms   will   be    destroyed   and   the   specific 
organism  may  be  isolated  in  pure  culture  from  diseased  tissue  or 
lesions  of  the  animal  so  infected.     The  animal  body  is  used  as 
a  kind  of  filter  for  the  removal  of  the  non-pathogenic  bacteria. 

4.  In   determining  the  strength   of   concentration   of   certain 
biological  products.     As  will  be  seen  later,  the  only  way  that  has 
been  devised  for  the  determination  of  the  strength  or  potency  of 
certain  poisons,  such  as  toxins,  and  for  their  antitoxins,  is  animal 
injection.     The  animal  is  used  by  the  bacteriologist  in  much  the 
same  manner  as  an  indicator  is  used  by  the  chemist  in  determining 
the  acidity  or  alkalinity  of  a  solution. 

5.  In  the  production  of  certain  so-called  antibodies,  such  as  anti- 
toxins, and  for  the  demonstration  of  certain  characteristics  of  the 
blood-serum  in  immunity. 

The  animal  most  frequently  used  in  experimental  work  is  the 


118  VETERINARY   BACTERIOLOGY 

guinea-pig  or  cavy;  next  in  importance  is  the  rabbit.  Mice  and 
rats,  particularly  the  white  varieties,  are  sometimes  used.  When 
birds  are  necessary,  the  pigeon  and  domestic  fowl  are  generally 
utilized.  Some  of  the  larger  animals,  as  the  goat  or  horse,  are 
used  for  the  production  of  serum,  where  it  is  required  in  con- 
siderable quantities,  as  in  the  manufacture  of  antitoxins.  The 
monkey  has  been  used  to  some  extent  in  the  study  of  diseases 
peculiar  to  man.  Heifers  are  utilized  in  the  preparation  of  the 
vaccine  against  small-pox.  Swine  are  used  in  the  preparation  of 
hog-cholera  antiserum. 

Methods  of  Inoculation. — Animals  are  commonly  inoculated  just 
beneath  the  skin  or  subcutaneously .  The  hair  is  shaved  from  the 
area  selected,  the  skin  is  washed  with  an  antiseptic,  and  a  hypo- 
dermic needle  inserted  into  the  subcutaneous  tissue.  In  the  inocu- 


Fig.  64. — Ear  veins  of  a  rabbit:  a,  Posterior  vein;  6,  point  at  which  injections 
may  be  most  easily  made;  c,  median  vein  (adapted  from  Frost). 

lation  of  a  solid  material  a  little  incision  may  be  made  in  the  skin, 
the  material  inserted,  and  the  flaps  of  skin  pulled  together.  Usu- 
ally stitches  to  hold  the  skin  are  unnecessary.  Intravenous 
inoculation  is  accomplished  by  inserting  the  needle  into  a  vein. 
Usually  a  rabbit  is  selected  for  this  purpose.  Reference  to  Fig. 
64  will  show  the  vein  on  the  posterior  edge  of  the  ear,  into  which 
injections  are  usually  made.  The  large  median  vein  is  not  suitable, 
as  it  is  situated  in  loose  connective  tissue  and  therefore  difficult 
to  enter.  The  posterior  vein,  on  the  other  hand,  is  embedded  in 
firm  connective  tissue  and  cartilage,  so  that  it  does  not  give  before 
the  needle-point. 

Intraperitoneal  injection  is  accomplished  by  thrusting  the 
hypodermic  needle  through  the  abdominal  wall.  Some  care  must 
be  used  not  to  penetrate  too  rapidly,  as  there  is  danger  of  injuring 


BACTERIA   AND    DISEASE;    GENERAL   CONSIDERATIONS       119 

the  intestines.  Intrathoracic  inoculation  is  rarely  practised. 
Injection  directly  into  the  heart  (intracardiac)  may  be  successfully 
practised  if  sufficient  care  is  used.  Inoculation  by  scarification 
is  accomplished  by  scraping  off  the  outer  layers  of  skin  without 
drawing  blood  and  rubbing  the  organism  on  the  surface  moistened 
by  the  exuded  serum.  Intracranial  injections  may  be  made  by  the 
use  of  a  trephine  to  penetrate  the  skull,  when  subdural  inocula- 
tions are  made  with  the  hypodermic.  Intra-ocular  injections 
have  sometimes  been  performed  for  the  specific  purpose  of  observ- 
ing from  day  to  day  the  development  of  lesions  upon  the  iris 
or  in  other  tissues  of  the  eye.  Inoculation  by  inhalation  is  accomp- 
lished by  forcing  the  animal  to  breathe  the  organism  in  dust  or  as 
a  fine  spray.  Infection  by  way  of  the  digestive  tract  is  accomp- 
lished by  feeding  or  ingestion. 

Interrelationships  of  the  Organism  and  the  Body. — Any  organ- 
ism which  produces  lesions  or  morbid  changes  of  any  kind  in  the 
body  is  said  to  be  pathogenic,  or  disease  producing.  By  virulence 
is  meant  the  relative  ability  of  organisms  of  different  races  to 
produce  disease;  for  example,  one  culture  of  the  diphtheria  bacillus 
might  produce  a  severe  type  of  disease,  while  another  might  be 
wholly  unable  to  produce  an  infection  except  under  the  most  favor- 
able conditions.  The  former  is  said  to  be  more  virulent  than  the 
latter. 

Effects  of  Pathogenic  Bacteria  on  the  Body. — When  bacteria  infect 
the  body,  they  may  remain  at  or  near  the  site  of  infection;  they 
may  spread  through  the  tissues  by  direct  growth,  or  be  carried  by 
the  lymph  and  blood  to  other  parts  of  the  body;  they  may  multiply 
in  the  blood  or  they  may  produce  metastatic  infections  by  becoming 
localized  in  other  parts  of  the  body.  Disease  may  be  produced 
by  the  action  of  poisons,  either  toxins  or  endotoxins,  or  possibly 
mechanically.  The  following  classification  adapted  from  Muir 
and  Ritchie  is  useful  in  a  consideration  of  the  types  of  changes 
brought  about  in  the  body  by  microorganisms. 

A.  Tissue  changes. 

1.  Produced  in  the  immediate  vicinity  of  the  bacteria,  either 

at  the  primary  lesion  or  at  secondary  foci, 
(a)    Those    changes   resulting   directly .  from   damage,    as 
degeneration  and  necrosis. 


120  VETERINARY    BACTERIOLOGY 

(6)  Those  of  a  defensive  or  reparative  nature,  as  production 
of  new  tissue  and  invasion  of  lesion  by  phagocytic 
leukocytes. 

(1)  Increased  functional  activity. 

(2)  Increased  formative  activity,  cell  growth,  and  division. 
2.  Produced  at  a  distance  from  the  bacteria,  due  directly  or 

indirectly   to    poisonous    substances    produced    by    the 
bacteria, 
(a)  In  special  tissues. 

(1)  Changes  resulting  from  damage. 

(2)  Changes  of  a  reactive  nature  in  blood-forming  organs. 
(6)  General  anatomical  changes,  effects  of  malnutrition  or 

of  increased  waste. 
B.  Changes  in  metabolism,  fever,  etc. 


CHAPTER  XIII 

IMMUNITY,    GENERAL  DISCUSSION 

Immunity. — Immunity  is  a  term  used  to  express  relative  resist- 
ance to  disease.  It  is  denned  by  Ricketts  as  follows:  "  By  immun- 
ity we  understand  that  condition  in  which  an  individual  or  a  species 
of  animals  exhibits  unusual  or  complete  resistance  to  an  infection 
for  which  other  individuals  or  other  species  show  a  greater  or  less 
degree  of  susceptibility."  The  converse  of  immunity  is  suscepti- 
bility, or  lack  of  resistance. 

Resistance  by  the  body  to  infection  by  microorganisms  is 
due  to  a  considerable  number  of  factors.  These  may  be  grouped 
into  two  classes — the  external  resistance,  due  to  body  coverings 
and  protective  devices,  and  internal  resistance,  due  to  tissue  and 
body  humor  reactions. 

External  resistance  to  infection  is  of  the  greatest  practical 
importance.  The  skin  covering  the  surface  of  the  body  is  an  excel- 
lent and  effective  barrier  against  bacterial  invasion.  The  skin 
is  constantly  sloughing  off  at  the  surface  and  being  replaced  from 
below.  Entrance  to  the  tissues  is  sometimes  effected  by  micro- 
organisms through  the  hair-follicles  and  the  sweat-glands;  this  is, 
however,  exceptional.  The  subcutaneous  tissues  likewise  obstruct 
the  inward  growth  of  organisms  that  have  penetrated  the  skin. 
The  mucous  membranes  constantly  secrete  mucus,  which  is  as  con- 
stantly removed,  and  the  bacteria  which  have  been  caught  go  with 
it.  The  membranes  which  line  the  air-passages  catch  upon  their 
moist  surfaces  practically  all  the  bacteria  that  enter  with  the 
inspired  air,  and  few  ever  reach  the  ultimate  ramifications  of  the 
bronchioles,  much  less  the  alveoli.  The  gastric  juice  of  the  stomach 
is  markedly  germicidal.  Many,  though  not  all,  bacteria  which 
enter  the  body  with  the  food  are  destroyed  there.  The  intestinal 
juices,  particularly  the  bile,  are  mildly  antiseptic  and  inhibit  the 
growth  of  many  forms,  although  they  are  without  effect  on  others, 
among  them  the  so-called  normal  intestinal  bacteria. 

121 


122  VETERINARY   BACTERIOLOGY 

Variation  of  Individuals  in  Susceptibility  to  Disease.  Pre- 
disposing Factors. — The  same  individual  varies  greatly  at  times 
in  his  ability  to  resist  infection  by  bacteria.  Age  frequently  deter- 
mines resistance  to  certain  infections.  For  example,  there  are 
diseases,  such  as  diphtheria  and  whooping-cough,  which  are 
much  more  common  among  children  than  among  adults.  Black- 
leg rarely  attacks  adult  cattle.  Advancing  age  seems  to  bring 
increased  resistance  to  such  infections.  The  mechanism  of  this 
kind  of  resistance  to  infection  is  not  well  understood.  Hunger 
and  thirst  reduce  the  resistance  of  the  body  and  predispose  to 
infection.  Exposure  to  excessive  heat  or  the  chilling  of  the  body 
surfaces  by  cold  will  also  reduce  the  body  resistance  so  that  organ- 
isms that  ordinarily  cannot  produce  disease  gain  a  foothold. 
Fatigue  has  been  demonstrated  experimentally  to  render  animals 
and  man  more  susceptible  to  infection.  The  classic  example  of 
this  diminution  of  resistance  by  fatigue  is  that  of  the  white  rat, 
which  is  normally  immune  to  anthrax,  but  which,  when  exhausted 
by  work  in  a  treadmill,  becomes  susceptible  to  the  disease  and  will 
succumb  to  infection. 

Types  of  Immunity. — Immunity  may  be  divided  into  two  types, 
natural  and  acquired.  The  former  may  be  subdivided  again  into 
racial  or  specific  immunity  and  individual  immunity.  Acquired 
immunity  may  be  either  active  or  passive. 

Natural  immunity  is  congenital,  that  is,  it  is  not  acquired  after 
birth.  A  racial  immunity  is  one  possessed  by  all  the  members  of 
a  group  of  individuals.  Disease  frequently  cannot  be  transmitted 
from  one  species  of  animal  to  another,  for  example,  man  does  not 
acquire  many  of  the  diseases  of  animals,  such  as  hog-cholera  and 
dog  distemper,  and,  on  the  other  hand,  many  diseases  of  man,  such 
as  measles,  whooping-cough,  and  typhoid  fever,  cannot  be  trans- 
mitted to  the  lower  animals.  It  is  also  said  that  the  Algerian  alone 
among  the  breeds  of  sheep  is  naturally  immune  to  anthrax.  In 
New  York  City  it  has  been  found  that  the  Russian  and  Polish 
Jews  are  much  more  resistant  to  tuberculosis  than  are  certain  other 
races,  particularly  the  Irish  and  the  negroes — at  least  the  death- 
rate  among  the  former  due  to  this  disease  is  much  lower.  Individ- 
uals are  also  found  who  are  naturally  immune  to  disease.  This  re- 
sistance is  perhaps  more  seeming  than  real  in  many  cases,  and  has 


IMMUNITY.      GENERAL  DISCUSSION  123 

been  induced  by  mild  infections  that  have  resulted  in  recovery 
without  the  development  of  definite  symptoms  of  the  disease. 
It  is  a  well-known  fact  that  frequently  a  small  percentage  of  the 
hogs  in  a  herd  which  becomes  infected  with  hog-cholera  do  not 
contract  the  disease,  and  inoculation  experiments  show  them  to  be 
relatively  immune. 

Acquired  Immunity. — Immunity  to  a  disease  may  be  acquired 
in  many  different  ways.  An  active  acquired  immunity  is  one  brought 
about  by  the  development  in  the  tissues  of  the  animal  of  certain 
antibodies  which  prevent  the  growth  or  destroy  or  neutralize  the 
products  of  growth  of  the  invading  microorganisms.  Passive 
acquired  immunity  is  conferred  upon  the  animal  by  the  injection 
of  antibodies  which  have  been  prepared  in  another  animal.  A 
passively  immunized  animal  takes  no  part  in  the  production  of 
the  antibodies  to  which  it  owes  its  immunity. 

Active  Acquired  Immunity. — A  development  of  antibodies 
and  the  consequent  immunization  of  an  animal  may  be  brought 
about  by  the  various  methods  illustrated  in  the  following  out- 
line: 

A.  Injection  of  living  microorganisms. 

1.  In  quantities  smaller  than  the  amount  needed  to  produce 

a  fatal  infection. 

2.  Attenuated  in  various  ways — 

(a)  By  growing  upon  artificial  culture-media. 
(6)  By  growing  at  unusual  temperatures. 

(c)  By  heating. 

(d)  By  growing  in  the  presence  of  substances  inimical  to 
growth,  such  as  weak  antiseptics. 

(e)  By  animal  passage. 

B.  By  injection  of  dead  organisms. 

C.  By  injection  of  the  products  of  autolytic  digestion  of  or- 
ganisms. 

D.  By  injection  of  toxins. 

Immunization  by  the  injection  of  sublethal  doses  of  pathogenic 
organisms  has  not  been  generally  practised.  With  most  pathogenic 
organisms  it  may  be  demonstrated  experimentally  that  if  the 
number  of  living  cells  injected  be  decreased  below  a  certain  mini- 
mum, they  are  no  longer  capable  of  producing  disease.  The  method 


124  VETERINARY   BACTERIOLOGY 

is  dangerous  because  the  minimum  may  vary  with  different 
individuals  and  the  animal  utilized  may  in  some  instances  prove 
susceptible  and  succumb.  Usually,  however,  increasing  numbers 
of  the  organism  may  be  given,  and  the  animal  will  eventually 
become  entirely  immune.  This  method  is  of  more  theoretical 
than  practical  importance,  and  is  rarely  used. 

As  noted  above,  microorganisms  may  be  attenuated,  that  is, 
their  ability  to  produce  disease  lessened  in  several  ways.  Several 
species  of  bacteria  are  known  gradually  to  lessen  their  virulence 
when  cultivated  for  a  time  upon  artificial  media;  for  example, 
the  Streptococcus  pyogenes,  which  produces  many  suppurative  in- 
fections, when  cultivated  upon  artificial  media,  will  finally  lose 
so  much  of  its  virulence  that  it  proves  entirely  non-pathogenic 
when  inoculated  into  suitable  animals.  Inoculation  of  such  non- 
virulent  types  will  increase  the  resistance  of  the  body  to  the  viru- 
lent forms.  The  converse  of  this  is  also  true,  for  the  continued 
growth  and  transfer  of  many  organisms  from  one  animal  to  another 
may  greatly  exalt  the  virulence.  It  is  possible  in  a  given  species 
to  secure  in  some  cases  every  gradation  from  the  wholly  non- 
pathogenic  type  to  forms  that  are  exceptionally  virulent. 

Cultivation  of  some  bacteria  at  a  temperature  higher  than 
the  growth  optimum  results  in  a  diminution  of  virulence.  The 
anthrax  bacillus,  whose  optimum  is  38°,  may  be  cultivated  at  a 
temperature  of  "42°  for  a  time,  when  it  loses  much  of  its  virulence. 
It  may  then  be  used  in  the  form  of  injections  to  increase  resistance 
to  the  disease  anthrax  in  animals.  The  organism  which  causes 
blackleg  in  cattle  is  heated  to  a  temperature  just  below  its  thermal 
death-point,  and  is  found  to  lose  many  of  its  pathogenic  proper- 
ties, although  it  still  causes  the  production  of  immune  substances 
when  injected  into  the  body.  A  closely  related  method  is  to  grow 
bacteria  in  the  presence  of  mild  antiseptics.  The  anthrax  bacillus 
grown  in  the  presence  of  carbolic  acid  (1:  600)  has  been  found  to 
lose  its  virulence.  The  growth  of  certain  microorganisms  in  the 
body  of  animals  other  than  the  species  in  which  they  normally 
produce  disease  in  some  cases  results  in  a  decrease  of  virulence 
for  the  first  species.  The  small-pox  organism,  for  example,  is 
grown  for  a  time  in  the  bodies  of  cattle,  and  is  found  to  lose  much 
of  its  virulence  for  man  by  this  means.  The  injection  of  dead 


IMMUNITY.      GENERAL   DISCUSSION  125 

microorganisms  also  results  in  conferring  immunity  in  much  the 
same  manner,  perhaps,  as  does  the  injection  of  non-virulent 
cultures.  The  injection  of  bacteria,  whether  dead  or  alive,  in  an 
effort  to  increase  the  immunity  of  the  body,  is  known  as  vaccina- 
tion. This  is  also  denned  as  the  production  of  an  infection  that 
will  run  a  benign  course.  Bacterial  cultures  are  sometimes 
allowed  to  stand  until  a  considerable  amount  of  digestion  or 
autolysis  has  occurred.  When  the  dead  or  living  bacteria  are 
filtered  out  by  means  of  porcelain  filters,  the  material  remaining 
in  solution  or  the  filtrate  may  be  used  for  animal  injection  in  an 
effort  to  confer  immunity.  In  other  cases  the  bacteria  are  grown 
in  solutions  where  they  produce  certain  specific  poisons  called 
toxins.  These  latter  are  removed  by  filtration  and  used  to  inject 
animals  to  induce  the  development  of  an  active  immunity. 

Acquired  Passive  Immunity. — Immunity  may  be  conferred  by 
the  injection  of  serum  that  contains  suitable  antibodies.  These 
may  be,  as  will  be  seen  later,  in  the  nature  of  antitoxins,  bac- 
teriolysins,  or  opsonins.  This  method  of  conferring  immunity 
should  not  be  confused  with  vaccination.  The  animal  passively 
immunized  takes  absolutely  no  part  in  the  development  of  its 
immunity. 

Theories  of  Immunity. — Four  principal  theories  of  immunity 
have  been  held  since  the  acceptance  of  the  germ  theory  of  disease. 
The  first  two  proposed  are  now  of  historic  interest  only,  but  the 
other  two  are  well  founded  on  fact  and  are  generally  accepted 
at  the  present  time.  When  first  proposed,  these  latter  were  sup- 
posed to  be  antagonistic,  but  as  subsequently  modified,  they 
have  been  found  to  supplement  each  other,  some  facts  being 
explained  by  the  one  and  some  by  the  other.  These  theories 
are  worthy  of  brief  consideration  and  comparison. 

Theory  of  Exhaustion. — It  was  noted  by  early  laboratory 
workers  that  bacteria,  yeasts,  and  molds  would  grow  rapidly 
when  first  planted  upon  a  favorable  medium,  then  the  rate  of 
growth  would  become  slower,  and  finally  cease.  It  was  concluded 
naturally  that  growth  stopped  because  all  of  certain  of  the  nutrients 
needed  were  exhausted.  This  theory  was  applied  to  the  growth  of 
microorganisms  in  the  body,  and  it  was  believed  that  immunity 
was  established  when  certain  requisite  food  materials  were  ex- 


126  VETERINARY   BACTERIOLOGY 

hausted  and  the  organism  in  consequence  could  grow  no  longer. 
This  theory  was  soon  disproved.  It  was  shown,  for  example,  that 
the  Bacillus  diphtheria  would  grow  luxuriantly  on  sterilized  blood 
taken  from  a  person  immune  to  diphtheria.  Chemical  analyses 
also  showed  that  not  all  nutrients  were  exhausted  from  the  medium 
in  the  culture  tube  when  the  organism  ceased  growing. 

Noxious  Retention  Theory. — Further  study  revealed  the  fact 
that  organisms  ceased  growing  in  a  culture  solution  because  they 
produced  substances  deleterious  to  themselves.  The  organism 
which  sours  milk,  for  example,  produces  acid  until  the  concen- 
tration is  so  great  that  it  can  no  longer  develop.  The  same  is 
true  of  yeasts  in  the  production  of  alcohol,  and 'of  bacteria  in  the 
transformation  of  alcohol  to  acetic  acid.  The  nature  of  the 
noxious  material  is  known  in  very  few  cases.  A  logical  explana- 
tion of  immunity  seemed  to  be  that  something  of  the  same 
kind  occurred  in  acquired  immunity;  that  the  organisms  devel- 
oped in  the  body  until  they  produced  so  much  material  inimical 
to  their  own  growth  that  multiplication  would  cease,  and  the 
individual  would  thereafter  be  immune.  This  theory  was  dis- 
credited by  subsequent  workers,  who  proved  that  the  substances 
that  prevented  bacterial  growth  were  produced  by  the  body  and 
not  by  the  bacteria. 

Metchnikoff's  Theory  of  Phagocytosis. — The  theory  was 
advanced  by  Metchnikoff  and  his  students  that  certain  of  the 
body-cells,  particularly  the  leukocytes,  would  take  up,  digest,  and 
destroy  bacteria.  Immunization  accordingly  consisted  in  a  kind 
of  stimulation  or  training  of  the  leukocytes  to  destroy  the  patho- 
genic organisms.  Cells  capable  of  destroying  microorganisms  in 
this  manner  he  called  phagocytes  (Gr.  phagein,  to  eat;  kytos,  cell). 
Certain  of  Metchnikoff's  ideas  have  not  borne  the  test  of  time,  but 
in  the  main  his  theory  still  is  used  to  account  for  immunity  of 
certain  types. 

Ehrlich's  Humoral  Theory. — Ehrlich  has  advanced  the  theory 
that  immunity  is  due  to  substances  present  in  the  body  humors 
which  antagonize  the  growth  and  development  of  pathogenic 
organisms  or  are  capable  of  neutralizing  their  products.  Such 
substances  capable  of  conferring  immunity  he  calls  antibodies. 
and  to  their  production  by  the  body-cells  he  attributes  the  devel- 


IMMUNITY.      GENERAL   DISCUSSION  127 

opment  of  immunity.  This  theory  has  been  tested  in  a  very 
great  variety  of  ways,  and  seems  to  explain  better  than  any  other 
most  of  the  facts  known  relative  to  immunity. 

Duration  of  Immunity. — Recovery  from  certain  diseases, 
such  as  small-pox,  confers  a  lasting  immunity ;  from  others,  such  as 
pneumonia,  a  temporary  immunity,  and  in  still  others  the  im- 
munity disappears  immediately  upon  recovery,  as  in  influenza. 
In  some  cases  recovery  from  an  attack  seems  actually  to  predis- 
pose to  a  recurrence,  as  in  erysipelas.  This  last  probably  means 
simply  the  complete  or  relatively  complete  disappearance  of  im- 
munity from  a  peculiarly  susceptible  individual.  The  duration 
of  immunity  may  depend  in  some  cases  upon  the  type  of  anti- 
bodies produced,  although  this  is  certainly  not  always  the  case. 
Probably  the  antibodies,  particularly  those  introduced  in  passive 
immunization,  are  eliminated  in  some  of  the  secretions  and  ex- 
cretions, or  they  may,  in  some  cases,  be  easily  destroyed. 

Antigens  and  Antibodies. — It  has  been  found  experimentally 
that  injections  of  many  substances  besides  bacteria  and  their 
products  cause  reactions  on  the  part  of  the  tissues,  with  conse- 
quent production  of  antibodies.  These  substances  are  called  anti- 
gens. An  antigen  is,  therefore,  that  substance  which,  when  intro- 
duced into  the  body,  stimulates  the  tissues  to  the  production  of 
antibodies.  An  antibody  may  be  more  accurately  defined  as  any 
substance  present  in  the  serum  which  is  capable  of  neutralizing, 
antagonizing,  precipitating,  agglutinating,  or  dissolving  the  sub- 
stance (antigen)  which  has  induced  the  formation  of  such  anti- 
body. For  example,  the  toxin  of  the  diphtheria  bacillus,  when 
injected  in  non-lethal  doses,  induces  the  production  of  antitoxin 
by  the  tissues :  the.fiwA8  is  the  antigen,  the  antitoxin,  the  anti- 
body. Similarly,  egg-white  injected  mto  an  animal  would  be 
termed  an  antigen  and  the  precipitating  substance  produced  as 
a  consequence  in  the  blood-serum  is  termed  the  antibody. 

Antibodies  as  Factors  in  Acquired  Immunity. — The  somatic 
reactions  to  the  presence  of  antigens  are  now  generally  considered 
of  primary  importance  in  acquired  immunity.  Certain  bacterial 
poisons  called  toxins  cause  the  production  by  the  animal  tissues 
of  the  antibody  called  antitoxin,  which  will  neutralize  the  toxin. 
The  presence  of  certain  bacteria  in  the  body  causes  the  production 


128  VETERINARY    BACTERIOLOGY 

of  bacteriolysins,  substances  which  will  dissolve  or  destroy  bacteria. 
Opsonins  (Gr.  opsonein,  to  prepare  for  eating)  are  antibodies 
which  will  unite  with  the  bacteria  and  enable  the  phagocytes 
to  take  them  up  and  destroy  them.  Acquired  immunity  may, 
therefore,  be  said  to  be  either  antitoxic,  bacteriolytic  (antibacterial)., 
or  opsonic,  or  a  combination  of  any  or  all  of  these  types.  Three 
other  types  of  body  reactions  are  also  generally  considered  in  a 
discussion  of  immunity.  Although  they  probably  in  no  instance 
actually  account  for  immunity,  they  are  found  very  useful  in 
diagnosis,  and  their  consideration  is  quite  essential  to  a  full  dis- 
cussion of  theories  of  immunity.  The  blood-serum  of  individuals 
suffering  from  certain  infections,  particularly  bacterial,  acquires 
the  property  of  agglutinating  or  clumping  these  organisms.  The 
blood-serum  from  a  horse  having  glanders  when  dropped  into  a 
culture  of  the  glanders  bacillus  will  cause  the  bacteria  to  clump 
together  and  settle  out,  leaving  the  medium  clear.  The  phenom- 
enon of  agglutination  is  used  in  the  diagnosis  of  this  and  other 
diseases.  A  somewhat  similar  body  reaction  is  provoked  by  the 
injection  of  soluble  proteins  derived  from  some  other  species  of 
animal  (or  plant)  into  the  body  of  an  animal.  The  serum  of  such 
an  animal  acquires  the  property  of  precipitating  the  corresponding 
protein  when  mixed  with  it  in  a  test-tube.  This  phenomenon 
of  precipitation  is  made  use  of  in  the  differentiation  of  many  kinds 
of  proteins.  The  presence  of  certain  substances  in  the  blood, 
usually  proteins  derived  from  any  foreign  source,  may  result  in 
the  sensitization  of  the  body  to  such,  rather  than  immunity.  This 
phenomenon  is  known  as  anaphylaxis  (Gr.  an,  against;  phylasco, 
to  guard)  and  has  led  to  an  explanation  of  many  otherwise  obscure 
pathological  and  bacteriological  facts. 


CHAPTER  XIV 
ANTITOXINS  AND  RELATED  ANTIBODIES 

(Antibodies  of  Ehrlich's  First  Order) 

Toxins. — The  word  toxin  has  been  used  with  a  considerable 
variety  of  meanings.  A  substance  is  said  to  be  toxic  when  it 
brings  about  an  abnormal  condition  when  introduced  into  the 
body.  The  discovery  that  certain  microorganisms,  particularly 
bacteria,  produced  their  harmful  effects  by  means  of  the  poisons 
which  they  excreted  led  to  the  use  of  the  term  toxin  to  include  all 
poisons  produced  by  bacteria.  Further  study  demonstrated  that 
these  bacterial  poisons  differed  considerably  in  their  method 
of  action  and  in  other  characters.  Ehrlich  first  clearly  differen- 
tiated the  bacterial  poisons  and  used  the  name  toxin  to  indicate 
a  definite  type.  The  name  endotoxin  is  used  to  designate  certain 
poisonous  substances  that  are  contained  within  the  protoplasm 
of  the  cell,  and  are  not  excreted  as  are  the  true  toxins.  Some 
authors  have  included  all  these  poisonous  substances  produced 
by  bacteria  under  the  name  toxine,  and  recognize  toxin  as  used  in 
Ehrlich's  sense. 

Characteristics  of  a  Toxin  (Ehrlich). — According  to  Ehrlich, 
the  toxins  constitute  a  group  of  substances  having  the  following 
-  characteristics : 

1.  The  true  toxins  are  labile,  that  is,  they  are  easily  destroyed 
by  heat,  by  acids,  by  exposure  to  light  and  air. 

2.  The  chemical  nature  of  the  toxins,  with  one  possible  excep- 
tion, is  not  understood.     Beyond  the  fact  that  they  are  organic 
in  origin  and  usually  give  some  of  the  protein  reactions,  little  is 
known  of  their  chemistry. 

3.  Biological  tests  (i.  e.,  animal  inoculations)  have  been  found 
to  be  the  only  tests  by  which  the  toxins  may  be  recognized  and 
studied.     These  animal  inoculations,  as  has  been  before  stated, 
are  quite  as  essential  in  determining  the  character  and  strength 

9  129 


130  VETERINARY   BACTERIOLOGY 

of  a  toxin  as  are  indicators  to  the  chemist  in  his  study  of  end- 
reactions. 

4.  Toxins  act  upon  the  body  by  combining  chemically  with 
definite  cells  and  tissues.     This  union  is  not  effected  at  once 
in  all  cases.     The  evidences  of  damage,  with  the  clinical  symp- 
toms of  poisoning,  do  not  appear  until  after  the  lapse  of  a  period 
of  incubation.     The  length  of  this  period  varies  with  different 
toxins — with  some  of  the  snake  venoms  it  is  very  short,  in  other 
toxins  it  is  a  matter  of  hours,  or  even  days. 

5.  The   injection  of  non-lethal  doses  of  toxin  into  a  suitable 
animal    causes  the  tissues   to   react   and   to   produce   antitoxin, 
which  will  neutralize  the  toxin,  and  result  in  immunization. 

Toxins  may  be  differentiated  from  most  other  poisonous  sub- 
stances with  which  they  may  be  confused  by  reference  to  the 
preceding.  Poisonous  alkaloids,  such  as  strychnin,  for  example, 
do  not  cause  the  production  of  antibodies.  Immunization  against 
a  toxin  is,  therefore,  not  to  be  confused  with  drug  habituation. 

Sources  of  Toxins. — Toxins  have  been  found  to  be  produced 
by  a  considerable  number  of  plants  and  animals.  Certain  of  the 
flowering  plants  form  powerful  toxins,  such  as  ricin  in  the  castor- 
oil  bean  (Ricinus  communis  and  R.  zanzibarensis) ,  abrin  from  the 
jequirity  bean  (Abrus  precatorius) ,  and  robin  from  the  bark  of  the 
locust  (Robinia  sp.).  The  pollen  of  certain  plants,  particularly 
certain  of  the  grasses,  the  golden-rod  and  rag-weeds,  is  poisonous 
to  some  individuals,  producing  hay-fever,  and  as  specific  antitox- 
ins have  been  prepared  for  them,  the  poison  must  be  regarded  as  a 
true  toxin.  Among  the  fungi,  certain  poisonous  mushrooms  (or 
"  toadstools  "),  as  the  Amanita,  have  been  shown  to  contain  toxins. 
Certain  molds  are  stated  to  produce  toxins,  particularly  Asper- 
gillus.  Toxins  have  been  demonstrated  in  the  animal  kingdom 
in  the  venom  of  snakes,  scorpions,  and  spiders,  in  the  skin  of  certain 
reptiles,  in  the  blood  of  the  eel  and  of  certain  fish.  A  few  species 
of  bacteria  have  been  shown  to  produce  toxins,  but  in  several  cases 
the  amount  and  character  of  the  toxin  are  relatively  unimportant. 
The  bacteria  which  have  been  found  to  form  appreciable  amounts 
of  toxin,  and  which  produce  lesions  of  the  body  tissues  through 
the  action  of  these  toxins,  are  relatively  few.  The  more  im- 
portant are  the  following: 


ANTITOXIN'S    AND    RELATED    ANTIBODIES  131 

Bacillus  diphtheria,  the  cause  of  diphtheria. 

Bacillus  tetani,  the  cause  of  tetanus  or  lockjaw. 

Bacillus  botulinus,  the  cause  of  certain  cases  of  botulism  or 
meat-poisoning. 

Bacillus  enteritidis,  found  in  certain  cases  of  meat-poisoning. 

The  organisms  in  which  true  toxins  have  been  demonstrated, 
but  in  which  the  toxin  production  does  not  seem  to  account  for 
the  lesions  of  the  disease,  are — 

Bacillus  pyocyaneus,  associated  with  suppurative  processes. 

Micrococcus  aureus,  associated  with  suppurative  processes. 

Micrococcus  albus,  associated  with  suppurative  processes. 

It  should  again  be  emphasized  that  the  above  list  of  bacteria 
does  not  include  all  those  which  may  produce  poisoning,  but 
does  include  the  most  important  known  to  produce  true  toxins. 
Many  other  species  are  known  to  produce  endotoxins. 

Specificity  of  Toxins. — Toxins  must  combine  with  the  cells 
or  tissues  of  the  body  in  order  to  injure  them.  Toxins  do  not  all 
attack  the  same  tissue,  but  show  a  selective  action.  In  some  cases 
a  number  of  tissues  may  be  injured,  in  others  the  damage  is 
limited  quite  strictly  to  one  type.  The  toxin  produced  by  the 
bacillus  of  tetanus  attacks  the  cells  of  the  central  nervous  system 
and  is  called  a  neurotoxin.  One  of  the  toxins  commonly  present 
in  snake  venom  destroys  red  blood-cells  (hemotoxiri) .  Probably 
some  animals  owe  their  immunity  to  certain  toxins  to  the  fact 
that  some  of  the  less  important  or  non-vital  body-tissues  will 
combine  with  the  toxin  and  prevent  its  union  with  more  vital 
portions. 

Antitoxins. — Antitoxins  are  antibodies  produced  by  the  tissues 
of  the  body  as  a  result  of  injection  or  presence  of  toxins.  The 
fact  of  antitoxin  production  may  be  readily  demonstrated  by  mix- 
ing the  serum  of  an  immune  animal  with  toxin  and  injecting  the 
mixture  into  a  suitable  animal.  The  normal  toxic  action  will 
be  found  to  be  inhibited  by  the  antitoxin  of  the  serum.  The 
most  generally  accepted  and  valuable  of  the  explanations  of  the 
production  of  antitoxins  by  the  body  is  that  offered  by  Ehrlich, 
based  upon  his  theory  of  cell  nutrition,  the  lateral  chain  or  side- 
chain  theory  of  immunity. 

Ehrlich's  Theory  of  Cell  Nutrition. — Several  explanations  have 


132  VETERINARY   BACTERIOLOGY 

been  offered  by  physiologists  for  the  phenomenon  of  cell  nutrition. 
Food  substances  carried  to  the  cell  by  the  blood  must  pass  through 
the  vessel  and  cell-walls  and  be  anchored  there  if  they  are  to  be 
used  in  any  of  the  processes  of  cell  metabolism.  According  to 
Ehrlich,  the  protoplasm  must  be  made  up  of  molecules  having  an 
affinity  for  a  great  variety  of  food  materials.  These  molecules 
he  conceives  to  be  made  up  of  a  central  portion  surrounded  by 
atomic  groups  which  unite  with  certain  food  molecules  and  bind 
them  to  the  cell.  These  atomic  groups  have  affinity  for  certain 
food  substances,  and,  therefore,  are  differently  constituted.  These 
atomic  groups  he  calls  side-chains  or,  better,  cell  receptors.  The 
character  of  these  receptors  may  be  illustrated  by  a  chemical 
analogy.  Benzene  has  the  following  formula: 

H  H 


H—  C  C—  H 

\        / 

c=c 
/     \ 

H  H 

Any  one  of  these  hydrogens  may  be  substituted  by  some  element 
or  group.  Suppose  one  such  to  be  replaced  by  the  carboxyl 
group  (COOH),  another  by  the  amino  group  (NH2),  another  by 
the  aldehyd  group  (CHO),  and  still  another  by  a  hydroxyl 
group  (OH).  Such  a  hypothetical  compound  might  be  illustrated 

as  follows: 

H  COOH 

W 

s    % 

OH—  C  C—  NH2 


/     \ 

H  CHO 

There  are  several  possible  ways  in  which  such  a  compound 
might  react.  Should  an  alkali  be  brought  into  contact  with  it, 
the  base  would  be  taken  up  by  the  carboxyl  atom  group.  Acids 
would  be  bound  by  the  amino  group.  Other  substances  wouM  be 
bound  by  other  atom  groups.  The  cell  receptors  must  be  con- 


ANTITOXINS   AND    RELATED   ANTIBODIES  133 

sidered  in  this  manner,  as  being  of  different  types,  each  one  capable 
of  uniting  with  some  food  substance — probably  only  one.  To  take 
up  the  variety  of  substances  necessary  for  cell  life  and  activity, 
it  is  evidently  necessary  that  there  should  be  a  multiplicity  of 
these  receptors,  and  their  action  must  be  considered  as  very  specific. 

It  is  found  convenient  to  represent  these  cell  receptors  in  a 
diagrammatic  form.  Such  is  scarcely  necessary  in  a  consideration 
of  antitoxin,  but  will  be  found  very  helpful  in  a  discussion  of  the 
more  complex  antibodies. 

Ehrlich's  Theory  of  Antitoxin  Production. — As  has  been  stated 
previously,  toxins  are  believed  to  combine  with  the  tissue-cells 
before  the  latter  can  be  injured.  This  union  of  toxin  with  the 
cell  takes  place  through  the  medium  of  the  cell  receptors,  some  of 
which  are  thus  diverted  from  their  normal  functions.  As  a  result, 
if  the  cell  is  not  too  seriously  injured  by  the  toxin,  it  or  the  neigh- 
boring cells  produce  an  increased  number  of  receptors  of  the  type 
thus  used.  This  is  in  accordance  with  the  general  hypothesis 
enunciated  by  Weigert,  that  injury  or  irritation  always  results  in 
an  overgrowth  of  tissue — a  hypercompensation.  For  example, 
rubbing  the  skin  will  produce  a  callus,  leaky  heart  valves  cause 
hypertrophy  of  the  heart,  and  the  cicatricial  tissue  of  a  newly 
healed  wound  is  generally  greater  in  amount  than  the  tissue  it 
replaces.  These  receptors  are,  therefore,  produced  in  great 
numbers,  and  many  are  eventually  displaced  and  escape  into  the 
cell-plasma  and  finally  into  the  blood-stream.  These  freed  cell 
receptors  still  retain  their  affinity  for  the  toxin  and  constitute  the 
antitoxin  molecules  of  the  serum.  For  each  of  the  statements  just 
made  there  seems  to  be  a  good  proof.  That  the  toxin  actually 
unites  with  the  body-cells  has  been  shown  by  experiment,  for 
example,  the  brain  tissue  of  an  animal  mixed  with  tetanus  toxin 
will  absorb  the  latter  and  remove  it  from  the  solution,  so  that 
it  is  without  effect  when  injected  into  animals.  That  there  is  an 
actual  increase  in  the  number  of  cell  receptors  may  be  shown  by 
injecting  a  small  amount  of  toxin  into  an  animal,  and  before  anti- 
toxins have  appeared  in  the  blood,  injecting  more  toxin.  The 
response  to  the  second  injection  will  be  quicker  than  to  the  first, 
and  the  animal  will  succumb  to  what  is  not  ordinarily  a  fatal 
dose.  This  indicates  an  increased  power  of  fixation  for  the  toxin, 


134  VETERINARY   BACTERIOLOGY 

i.  e.,  an  increase  in  the  number  of  the  receptors.  The  appearance 
and  increase  in  the  freed  cell  receptors  or  antitoxins  in  the  blood 
shows  that  these  receptors  are  thrown  off  in  large  numbers.  The 
antitoxin  in  the  serum  unites  with  the  toxin  probably  in  much  the 
same  manner  as  an  acid  neutralizes  an  alkali.  The  principal 
difference  is  that  in  carrying  out  the  test,  animal  inoculations 
must  take  the  place  of  the  indicator  of  the  chemist.  The  union 
between  the  toxin  and  antitoxin  has  been  shown  to  obey  the  gen- 
eral laws  of  chemical  union. 

Constitution  of  the  Toxin. — Toxins  are  easily  destroyed  by 
heat,  chemicals,  light,  and  air.  It  will  be  shown  later  that  the 
loss  of  toxicity  does  not  result  from  a  total  destruction  of  the 
toxin  molecule,  for  it  still  is  able  to  unite  with  the  antitoxin. 
It  is  evident  that  the  toxin  molecule  is  made  up  of  two  parts — • 
a  thermostabile  portion,  which  unites  with  cell  receptors,  either 
in  the  cell  or  free  as  antitoxins,  called  the  haptophore,  or  binding 
group,  and  a  thermolabile  group,  called  the  toxophore,  which  causes 
the  cell  injury  after  union  by  means  of  the  haptophore  has  taken 
place.  When  the  toxophore  group  of  a  toxin  has  been  destroyed, 
that  which  remains  is  called  a  toxoid.  That  toxoids  exist  may  be 
demonstrated  in  two  ways.  The  injection  of  a  toxin  solution 
that  has  been  heated  to  56  °  for  half  an  hour  into  a  suitable  animal 
does  not  result  in  the  development  of  symptoms  of  poisoning,  but 
does  cause  the  production  of  antitoxin;  in  other  words,  the  toxoids 
retain  the  ability  to  unite  with  the  cell  receptors  and  to  bring 
about  their  increase  and  elimination  from  the  cell.  An  antitoxic 
serum  may  be  mixed  in  suitable  amounts  with  a  solution  of 
heated  toxin  (toxoid),  and  after  a  time  mixed  with  virulent  toxin, 
and  the  latter  will  be  found  to  remain  uncombined.  The  anti- 
toxin is  completely  neutralized  by  the  toxoid,  and  the  toxin 
added  subsequently  finds  no  antitoxin  free  with  which  it  can 
unite. 

Constitution  of  Antitoxin. — Antitoxin  is  more  stable  than  the 
toxin  in  most  cases.  However,  it  is  easily  destroyed  by  a  tempera- 
ture of  60°  if  sufficiently  prolonged.  There  is  no  reason  to  sup- 
pose that  it  is  made  up  of  more  than  one  group.  Inasmuch  as  it 
has  a  binding  group,  this  may  be  called  a  haptophore.  It  has  not 
as  yet  proved  possible  to  separate  antitoxins  from  serum  globulins; 


ANTITOXINS   AND    RELATED   ANTIBODIES 


135 


it  is  inferred,  therefore,  that  they  are  similarly  constituted.  Further 
light  is  needed  on  this  subject. 

Diagrammatic  Representation  of  Toxin  and  Antitoxins.  —  The 
preceding  facts  may  in  large  part  be  recorded  by  diagrams.  These 
are,  of  course,  arbitrary  in  shape  and  appearance,  but  are  helpful 
in  an  understanding  of  the  reaction.  The  diagrams  commonly 
used  for  this  purpose  are  given  in  Fig.  65. 

Preferential  Union  of  Toxins  with  Body-cells.  —  The  union  of 
antitoxin  with  toxin  occurs  apparently  as  readily  within  the 


A.  5. 

Fig.  65.  —  Diagrammatic  representation  of  toxins  and  of  antitoxin  pro- 
duction —  1,  Toxin  molecule:  a,  Haptophore;  6,  toxophore.  2.  Toxoid,  a 
toxin  molecule  that  has  lost  its  toxophore.  3,  Molecule  of  cell  protoplasm 
showing  the  union  of  the  toxin  molecule:  a,  Free  toxin;  6,  toxin  attached  to 
a  cell  receptor  c;  d,  other  cell  receptors.  4  illustrates  the  overproduction  of 
receptors  by  the  cell  and  their  elimination  (6)  into  the  blood-stream  as  anti- 
toxin molecules  (c).  5,  Neutralization  or  union  of  antitoxin  with  toxoid 
and  toxin. 


body  as  without.  When  toxins  are  injected  and  antitoxins  are 
present  in  the  circulation,  the  latter  will  commonly  unite  with 
the  former  and  prevent  union  with  the  body-cells.  In  some 
exceptional  conditions,  however,  this  is  shown  not  to  occur;  for 
some  reason  the  affinity  of  cell  receptors  still  united  to  the  cell 
seems  to  be  greater  than  those  free  (antitoxin).  In  other  words, 
the  tissues  seem  to  become  hypersensitive  to  the  presence  of  the 
toxin,  so  that  the  antitoxin  no  longer  protects.  Tissue  immunity 


136  VETERINARY   BACTERIOLOGY 

is,  therefore,  not  always  the  same  as  antitoxin  immunity.  This 
hypersensitiveness  is  of  unusual  occurrence.  An  animal  that  is 
being  immunized  against  tetanus  toxin  by  injections  of  increasing 
amounts  may  suddenly  and  unexpectedly  show  a  marked  reaction, 
or  may  even  succumb,  when  additional  amounts  are  injected, 
even  though  antitoxin  may  be  demonstrated  in  considerable 
quantities  in  the  blood.  It  is  evident  that,  although  this  phe- 
nomenon is  of  infrequent  occurrence,  it  is  of  some  little  importance. 
Possibly  this  may  be  related  to  the  reactions  to  be  described  later 
under  the  heading  of  Anaphylaxis. 

Antitoxins  of  Commercial  Importance. — Antitoxins  specific 
for  the  venom  of  certain  snakes  and  for  diphtheria  and  tetanus 
toxins  are  prepared  commercially.  The  manufacture  and 
standardization  of  these  antitoxins  are  of  importance  to  the 
veterinarian  for  several  reasons.  The  theory  of  immunity  has 
been  largely  developed  through  a  study  of  diphtheria  toxins  and 
antitoxins.  The  larger  animals,  such  as  the  horse  and  goat,  are 
generally  used  in  the  commercial  manufacture  of  antitoxin. 
The  tetanus  antitoxin  is  used  extensively  in  veterinary  practice. 
A  brief  consideration  of  the  production  of  diphtheria  and  tetanus 
toxin  and  antitoxin  is,  therefore,  advisable. 

Manufacture  of  Diphtheria  Toxin  and  Antitoxin. — Diphtheria 
toxin  is  prepared  by  growing  the  diphtheria  bacillus  in  suitable 
broth.  A  sugar-free  broth  is  prepared  as  described  in  Chapter 
VII.  This  is  titrated  and  the  reaction  adjusted  accurately  to 
+0.5.  It  is  then  placed  in  flat-bottomed  toxin  flasks  in  a  layer  a 
few  centimeters  in  thickness,  and  autoclaved.  To  each  flask  is  then 
added  sufficient  sterile  dextrose  solution  to  make  a  0.2  per  cent, 
dextrose  broth.  It  is  found  in  experience  that  different  strains  of 
the  diphtheria  bacilli  may  vary  considerably  in  toxin  production. 
The  strain  used  most  frequently  in  the  United  States  is  the  one 
known  as  the  Park-Williams.  The  organism  is  grown  in  broth 
tubes,  where  it  forms  a  film  over  the  surface.  Portions  of  this  film 
are  transferred  carefully  to  the  surface  of  the  broth  in  the  flasks  and 
incubated  at  37°  for  eight  days.  Microscopic  examination  of  the 
culture  should  show  it  to  be  uncontaminated.  To  destroy  the 
bacteria  and  prevent  possible  infection  of  those  handling  the 
material  5  c.c.  of  phenol  or  similar  antiseptic  is  then  added  to 


ANTITOXINS   AND   RELATED   ANTIBODIES 


137 


each  liter.  In  some  cases  it  is  filtered  through  a  porcelain  filter, 
which  will  remove  the  bodies  of  the  bacteria,  but  not  the  soluble 
toxins.  The  amount  of  toxin  present  in  the  solution  must  be 
next  determined  for  two  reasons:  first,  to  insure  the  presence  of 
toxins  in  sufficient  concentration  for  efficient  immunization,  and, 
second,  to  determine  the  amount  that  may  be  injected  into  the 
horse  without  serious  injury.  The  amount  of  toxin  that,  when 
injected  subcutaneously,  will  kill  a  guinea-pig  weighing  250  gm. 
in  three  days  is  called  the  minimum  lethal  dose  for  the  guinea-pig 
(abbreviated  M.  L.  D.).  The  broth  should  contain  at  least  1000 


Fig.  66. — Apparatus  for  the  injection  of  considerable  quantities  of  toxin 
into  the  horse:  a,  Graduated  cylinder  closed  by  a  rubber  cork,  b.  Air  is 
pumped  into  the  constant  pressure  bulb  e,  and  from  this  passes  into  the  cylinder. 
The  toxin  is  forced  out  through  the  needle  at  i  (Levaditi). 


M.  L.  D.  per  cubic  centimeter.  The  horse  is  most  commonly 
employed  for  the  production  of  the  antitoxin.  Care  is  used  that 
the  animal  is  fairly  vigorous  and  entirely  free  from  any  infectious 
disease.  Injections  of  100  M.  L.  D.  are  made  subcutaneously, 
or,  better  yet,  larger  amounts,  neutralized  by  antitoxin  already 
prepared,  are  first  used.  The  animal  responds  by  fever  and 
swelling  at  point  of  injection  and  by  other  minor  symptoms. 
After  a  lapse  of  several  days,  or  when  the  horse  has  recovered,  a 


138 


VETERINARY   BACTERIOLOGY 


second  injection  of  a  larger  amount  is  given.  Increasing  doses  of 
the  toxin  are  injected  at  intervals  until  as  much  as  500  c.c.  of  the 
toxin  may  be  administered  at  one  time.  Not  all  horses  produce 
enough  antitoxin  in  their  blood  to  be  commercially  valuable; 
hence  the  antitoxin  content  is  usually  determined  some  time  be- 
fore the  process  of  immunization  is  complete.  When  the  animal's 
blood  contains  the  maximum  amount  of  antitoxin,  it  is  drawn 
by  a  sterile  trocar  from  the  right  jugular  vein  into  wide-mouthed 
sterile  jars  (Fig.  68).  Usually  a  little  less  than  one  liter  of  blood 
for  every  hundred  pounds  weight  of  the  horse  is  removed,  as  this 


Fig.  67. — Injection  of  a  horse  with  toxin  (Levaditi). 

amount  may  be  withdrawn  without  appreciable  injury.     After 
a  rest  the  horse  may  again  be  injected  and  bled. 

The  jars  of  blood  are  allowed  to  stand  until  the  clot  has  shrunk 
and  the  clear,  straw-colored  serum  has  separated.  This  contains 
the  antitoxin  and  is  the  serum  antidiphtheriticum  of  the  Pharma- 
copeia. It  must  now  be  standardized,  that  is,  the  amount  of 
antitoxin  per  cubic  centimeter  determined.  Several  methods 
of  determining  the  potency  of  the  antitoxin  have  been  pro- 
posed. Inasmuch  as  the  unit  formulated  by  Ehrlich  has  been 
generally  adopted  (except  by  the  French)  its  development  will 
be  briefly  traced.  Behring  first  proposed  as  an  antitoxic  unit, 


ANTITOXINS   AND    RELATED    ANTIBODIES  139 

the  least  amount  of  antitoxin  which,  when  mixed  with  100  M.  L. 
D.  of  the  toxin,  would  prevent  a  250-gm.  guinea-pig  injected  with 
the  mixture  from  dying  within  four  days.  This  implied  the  keep- 
ing of  the  toxin  of  a  certain  strength  for  the  standardization  of 
antitoxin.  The  toxin  was  found  to  be  unstable,  and  the  same 
toxin  would  at  different  times  yield  different  results.  Furthermore, 
antitoxins  are  neutralized  by  toxoids  as  well  as  toxins.  The  toxin 
cannot  be, preserved  for  long  periods  without  deterioration,  as 
would  be  necessitated  if  used  as  a  government  standard.  Ehrlich, 
therefore,  made  use  of  a  toxin  which  he  had  studied  in  his  labora- 
tory to  standardize  a  large  quantity  of  serum.  This  serum  he 
dried  in  a  current  of  warm  air  in  a  partial  vacuum.  As  a  result, 


Fig.  68. — A  trocar  inserted  into  the  jugular  vein  of  the  horse.  The  com- 
pressor causes  a  noticeable  engorgement  of  the  vessel  (Kretz  in  Kraus  and 
Levaditi). 

he  secured  a  considerable  amount  of  serum  in  the  form  of  dried 
scales.  The  number  of  immunity  units  (U.  I.)  per  gram  of  this 
dried  material  was  very  accurately  determined.  Exactly  equal 
amounts  by  weight  of  this  standard  serum  were  placed  in  each 
of  a  large  number  of  special  tubes.  The  serum  was  placed  in 
one  arm  and  phosphorus  pentoxid  (P2O5),  an  active  dehydrating 
agent,  in  the  other.  The  air  was  then  exhausted  as  completely 
as  possible  by  an  air-pump  and  the  tubes  sealed.  They  then  were 
placed  in  a  dark  refrigerator,  where  a  constant  temperature  was 
maintained.  The  antitoxin  under  these  conditions  was  found 
to  retain  its  potency  undiminished  for  a  long  period.  Ehrlich 


140 


VETERINARY    BACTERIOLOGY 


placed  1700  U.  I.  in  each  tube.  Each  month  a  tube  is  opened 
and  its  content  of  serum  dissolved  in  1700  c.c.  of  a  mixture  of 
water  and  glycerin.  Each  cubic  centimeter,  therefore,  contains  one 
immunity  unit  of  antitoxin.  A  careful  study  of  the  toxin  which 
Ehrlich  had  used  in  preparing  this  standard  showed  him  that  it 
contained,  in  addition  to  the  100  M.  L.  D.  of  toxin,  an  equal 
amount  of  toxoid.  Theoretically,  therefore,  this  immunity  unit 
prepared  by  him  contains  sufficient  antitoxin  to  neutralize  200 
M.  L.  D.  of  a  pure  toxin.  In  view  of  the  fact  that  all  antitoxin 


cotTon 


dr\e«j  serum 


Fig.  69. — A  tube  used  for  the  preservation  of  antitoxin  by  the  Hygienic 
Laboratory  of  the  Public  Health  and  Marine  Hospital  Service :  a,  Serum  scales, 
or  antitoxin;  6,  phosphorus  pentoxid,  an  active  dehydrating  agent  (M.  J. 
Rosenau,  Bulletin  No.  21,  Hygienic  Laboratory). 

in  this  country  is  now  standardized  with  reference  to  this  U.  I. 
of  Ehrlich,  the  immunity  unit  for  diphtheria  antitoxin  has  been 
redefined  as  an  amount  of  antitoxin  equivalent  to  that  contained 
in  1  c.c.  of  solution  when  the  contents  of  the  tubes  prepared  by 
Ehrlich  are  dissolved  in  1700  c.c.  of  water.  In  the  United  States 
a  similar  set  of  tubes  (Fig.  69)  has  been  prepared  by  the  Hygienic 
Laboratory  of  the  Public  Health  and  Marine  Hospital  Service, 
and  the  standard  serum  is  sent  from  that  laboratory  to  the  manu- 
facturers. 

The  old  antitoxin  cannot  be  used  directly  to  determine  the 
potency  of  new  antitoxin,  but  a  lot  of  toxin  must  first  be  standard- 
ized. It  is  necessary  to  express  the  strength  of  the  toxin  in  terms 


ANTITOXINS   AND   RELATED    ANTIBODIES 


141 


of  the  standard  antitoxin.  Toxin  is  used  which  has  been  pre- 
served until  the  first  rapid  transformation  of  toxin  to  toxoid  has 
ceased,  and  it  has,  therefore,  become  relatively  stable.  A  series 
of  syringe  tubes  is  prepared  (Fig.  70),  each  one  containing  one 
standard  immunity  unit  of  antitoxin,  and  to  these  are  added 
varying  amounts  of  the  toxin  to  be  tested;  each  is  then  injected 
into  a  250-gm.  guinea-pig.  The  amount  of  antitoxin  will  be  more 
than  sufficient  to  neutralize  the  toxin  in  some  cases,  and  the 
animals  injected  will  show  no  ill  effects;  in  other  cases  the  toxin 
will  be  in  excess,  and  the  animals  will  die.  The  amount  of  toxin 
which,  when  thus  mixed  with  1  U.  I.,  will  kill  a  250-gm.  guinea- 
pig  in  just  four  days  is  called  the  L+  dose.  The  amount  of  the 


Fig.  70. — Battery  of  syringe  tubas  for  testing  the  potency  of  toxin  and  anti- 
toxin (Madsen). 

toxin  solution  just  necessary  to  neutralize  an  immunity  unit,  as 
evidenced  by  the  almost  complete  lack  of  tissue  reaction  at  the 
site  of  injection,  is  called  the  LO  (limit  zero)  dose.  In  a  solution 
containing  toxins  only  (no  toxoids)  the  difference  between  the  L  + 
and  the  LO  dose  would  be  1  M.  L.  D.  of  the  toxin,  but  such  toxoid- 
free  solutions  cannot  be  obtained;  hence  the  difference  between 
the  two  is  greater.  The  only  reason  that  the  LO  dose  is  determined 
in  practice  is  that  a  toxin  solution,  in  which  the  L-f  and  LO  doses 
differ  widely,  is  not  suitable  for  carrying  out  the  test  and  should 
be  discarded.  When  the  L4-  dose  of  the  toxin  has  been  satis- 
factorily determined,  it  may  then  be  used  to  determine  the  potency 


142  VETERINARY   BACTERIOLOGY 

of  fresh  antitoxin.  A  series  of  syringe  tubes,  each  containing  one 
L-f  dose  of  the  toxin,  is  arranged,  and  the  varying  amounts  of 
the  antitoxin  serum  to  be  tested  are  added.  The  amount  of 
serum  which  will  prevent  a  250-gm.  guinea-pig  from  dying  in 
less  than  four  days  contains  1  immunity  unit. 

According  to  Ehrlich,  diphtheria  toxin  is  in  reality  made  up  of 
two  poisons — the  true  toxin  and  toxone.  The  latter  he  holds  to 
account  for  the  paralyses  that  are  frequent  sequelae  in  diphtheria. 
The  same  antitoxin  neutralizes  both  the  toxin  and  the  toxone. 
This  toxone  is  of  some  theoretic  and  practical  importance  in  the 
standardization  of  the  toxin  and  the  antitoxin. 

The  amount  of  antitoxin  present  varies  greatly  in  the  serum 
produced  by  different  horses.  One  which  contains  less  than 
250  U.  I.  per  cubic  centimeter  is  rarely  used.  The  greater  the 
concentration,  the  more  valuable  is  the  serum  for  prophylaxis  and 
cure.  Many  efforts  to  concentrate  diphtheria  antitoxin  have  been 
made,  all  based  upon  the  fact  that  the  blood-serum  is  a  mixture  of 
various  proteins,  and  that  the  antitoxin  seems  to  be  inseparably 
bound  up  with  certain  ones  only  of  these.  The  removal  of  the 
proteins  having  no  antitoxic  value  results  in  a  considerable  con- 
centration of  the  antitoxin-holding  proteins.  Several  methods 
have  been  devised  for  this  purpose.  All  are  based  upon  differences 
in  solubility  or  coagulability  of  the  various  serum  constituents. 
The  methods  of  Gibson  and  of  Banzhaf  deserve  mention.  The 
former  precipitates  the  serum  by  the  addition  of  an  equal  amount 
of  a  saturated  solution  of  ammonium  sulphate.  This  precipitates 
the  antitoxic  and  some  other  fractions  of  the  serum,  but  does  not 
precipitate  certain  of  the  non-antitoxic  albumins.  These  are 
filtered  out,  the  precipitate  is  dissolved  in  water  to  its  original 
volume,  and  is  again  precipitated  by  ammonium  sulphate.  This 
precipitate,  when  filtered,  is  relatively  free  from  albumins.  It 
is  then  stirred  into  a  saturated  solution  of  sodium  chlorid,  which 
dissolves  the  pseudoglobulins  with  the  antitoxin.  The  insoluble 
portions  are  filtered  out  and  the  clear  filtrate  acidified  by  the 
addition  of  0.25  per  cent,  of  80  per  cent,  acetic  acid.  The  pre- 
cipitate which  is  thrown  down  is  collected  over  hard  filters,  par- 
tially dried  by  pressure  with  bibulous  filter-paper,  and  placed  in  a 
parchment  bag  for  dialysis  in  running  water.  The  acid  is  neu- 


ANTITOXINS   AND    RELATED   ANTIBODIES 


143 


tralized  by  the  addition  of  sodium  carbonate,  and  dialysis  is  con- 
tinued until  the  soluble  salts  have  practically  disappeared.  If 
care  is  used,  the  antitoxin  will  be  found  to  be  dissolved  in  a  much 
smaller  quantity  of  water  than  originally  present  in  the  serum. 
The  concentration  may  be  from  two  to  three  and  one-half  times 
the  original. 

Banzhaf  makes  use  of  the  various  coagulation  temperatures 
of  the  serum  constituents  in  effecting  their  separation.  The 
albumins  and  part  of  the  non-antitoxic  globulins  are  precipitated 
by  heating  for  twenty-four  hours  at  58°.  Sodium  chlorid  crystals 
are  added  to  saturation,  and  much  of  the  remaining  globulin, 
transformed  by  heat,  is  precipitated,  leaving  the  pseudoglobulins 


Fig.  71. — One  type  of  nitration  apparatus  used  for  serum:  a,  Filter;  6, 
test-tube  within  a  filter  flask  from  which  the  air  is  partially  exhausted  by 
the  vacuum  pump  at  d  (Weidanz). 

and  the  associated  antitoxin  in  solution.  The  clear  filtrate  is 
acidified  with  acetic  acid,  and  the  precipitate  prepared  as  in  the 
preceding  method.  By  this  method  a  concentration  of  ten  times 
the  original  has  been  obtained. 

The  antitoxic  serum  is  in  all  cases  filtered  through  sterile  un- 
glazed  porcelain,  and,  after  the  addition  of  a  small  amount  of 
preservative,  placed  in  sterile  containers  and  sealed. 

Preparation  of  Tetanus  Toxin  and  Antitoxin. — The  tetanus 
toxin  is  prepared  by  growing  Bacillus  tetani  in  broth  under  anaerobic 
conditions.  This  may  be  accomplished  by  the  use  of  a  hj'drogen 


144 


VETERINARY   BACTERIOLOGY 


atmosphere,  but  more  easily  by  covering  the  medium  by  a  layer 
of  paraffin  or  other  neutral  oil.  The  methods  of  preparing  the 
toxin  for  use  and  of  manufacture  of  the  antitoxin  do  not  differ 
materially  from  those  used  in  the  production  of  diphtheria  anti- 
toxin. 

Unlike  diphtheria  toxin,  the  tetanus  toxin  may  be  dried  and 
preserved  indefinitely  without  deterioration.  It  is,  therefore,  the 
toxin  and  not  the  antitoxin  which  is  sent  out  from  the  Hygienic 


Fig.  72. — A  filtration  apparatus  after  Uhlenhuth  and  Weidanz. 

Laboratory  to  the  serum  laboratories  for  the  purpose  of  standard- 
ization. A  standard  toxin  has  been  prepared  at  this  laboratory, 
and  its  M.  L.  D.  for  a  350-gm.  guinea-pig  carefully  determined. 
This  is  sent  out  in  dried  form,  and  is  diluted  before  use,  so  that  each 
cubic  centimeter  contains  100  M.  L.  D.  of  the  toxin  for  a  350-gm. 
guinea-pig.  The  immunity  unit  is  defined  as  follows:  "  The 
immunity  unit  for  measuring  the  strength  of  tetanus  antitoxin 


ANTITOXINS   AND   RELATED    ANTIBODIES  145 

shall  be  ten  times  the  least  quantity  of  antitetanic  serum  neces- 
sary to  save  the  life  of  a  350-gm.  guinea-pig  for  ninety-six  hours, 
against  the  official  test-dose  of  a  standard  toxin  furnished  by  the 
Hygienic  Laboratory  of  the  Public  Health  and  Marine  Hospital 
Service."  1 

Another  statement  is  that  one-tenth  of  a  unit,  mixed  with 
100  M.  L.  D.  of  the  standard  toxin,  contains  "  just  enough  free 
poison  in  the  mixture  to  kill  the  guinea-pig  in  four  days  after 
subcutaneous  injection."  The  amount  of  toxoid  has  not  been 
accurately  determined  in  this  test-toxin  used;  therefore,  the  stand- 
ardization test  must  in  all  cases  be  in  terms  of  the  Hygienic  Labora- 
tory toxin,  no  other  sample  of  toxin  being  suitable.  The  test-dose 
is  called  the  L  +  dose,  as  in  the  diphtheria  toxin. 

Preparation  of  Other  Toxins  and  Antitoxins. — As  has  before 
been  stated,  antitoxins  have  been  prepared  for  a  large  number  of 
toxins.  The  two  already  discussed  are  by  far  of  the  greatest  im- 
portance commercially.  In  the  development  of  theories  of  im- 
munity considerable  use  has  been  made  of  antiricin  and  antiabrin. 
An  antitoxin  for  pollen  (called  pollantin)  has  been  used  to  some 
extent  in  hay-fever.  Antitoxins  against  snake  venom  may  be  pur- 
chased upon  the  market.  They  are  of  considerable  importance 
in  certain  tropical  countries,  particularly  India,  where  poisonous 
snakes  abound. 

Antienzymes. — A  study  of  enzymes  and  their  actions  has  shown 
them  to  resemble  toxins  in  some  respects.  Although  an  enzyme 
does  not  form  a  part  of  the  final  product  of  its  activity,  it  never- 
theless seems  evident  that  it  first  unites  with  the  compound  which 
it  transforms,  and  later  is  split  off.  Enzymes  are  believed  to 
possess  two  groups,  resembling  the  toxins — one  a  binding  group, 
or  haptophore,  and  the  other  a  fermenting  group,  or  zymophore. 
The  injection  of  an  enzyme  into  the  animal  body  will  usually 
result  in  the  production  of  an  antienzyme,  which  will  permanently 
combine  with  the  enzyme  and  effectually  prevent  its  action.  These 
antienzymes  are  probably  developed  in  exactly  the  same  manner 
as  are  the  antitoxins,  and  have  the  same  general  constitution. 
They  are  likewise  very  specific:  the  action  of  pepsin  is  inhibited 
by  an  antipepsin,  and  not  by  an  antirennet. 

1  Treasury  Dept.,  Circular  No.  61,  Oct.  25,  1907. 
10 


146  VETERINARY   BACTERIOLOGY 

Enzymes,  like  toxins,  are  thermolabile.  The  zymophore  may 
be  destroyed  without  injuring  the  haptophore. 

It  is  probable  that  there  are  certain  antiferments  constantly 
present  in  body  tissues  which  prevent  the  activity  of  the  autolytic 
and  other  enzymes  during  life. 

Other  Antibodies  Related  to  Antitoxins. — Antibodies  of  the 
same  type  as  the  antitoxin  have  been  produced  for  agglutinins, 
amboceptors,  complements,  and  other  bodies.  These  will  be 
considered  with  their  specific  antigens. 


CHAPTER  XV 
AGGLUTINATION  AND  PRECIPITATION 

(Antibodies  of  Ehrlich's  Second  Order) 

GRUBER,  in  1896,  discovered  that  the  blood  of  animals  immun- 
ized against  Bacillus  typhosus,  and  the  blood  of  patients  having  the 
disease,  when  added  to  a  liquid  culture  of  the  organism,  caused 
the  bacteria  to  cease  moving  and  to  clump  together.  This 
phenomenon  has  been  named  agglutination.  Later  it  was  found 
that  the  use  of  protein  substances  as  antigens  caused  the  pro- 
duction in  the  body  of.  substances  which,  when  mixed  with  the 
protein  in  solution,  would  form  a  precipitate.  This  is  known  as 
the  precipitation  phenomenon.  The  antibody  responsible  for 
agglutination  is  called  an  agglutinin;  for  precipitation,  a  predpitin. 

Differentiation  of  Precipitation  and  Agglutination. — The  dis- 
tinction between  agglutination  and  precipitation  may  be  stated  as 
follows:  Agglutination  occurs  when  the  antigen  is  in  suspension 
in  the  form  of  individual  cells  or  finely  divided  particles.  Pre- 
cipitation occurs  when  the  antigen  is  a  colloid  in  solution. 

Agglutination. — Agglutinins  may  be  grouped  into  two  classes, 
normal  and  immune.  A  normal  agglutinin  is  one  present  in  the 
body  without  any  infection  or  systematic  immunization.  An 
immune  agglutinin  is  one  that  is  developed  as  a  result  of  the  pres- 
ence of  an  organism  or  its  products  in  the  body.  There  is  no 
reason  to  suppose  that  the  normal  and  immune  agglutinins  differ 
from  each  other  in  any  essential  particular.  It  is  possible  that  all 
normal  agglutinins  are  in  reality  produced  as  a  result  of  an  unde- 
tected infection  or  to  the  presence  of  the  so-called  group  agglu- 
tinins to  be  considered  later. 

The  agglutination  reaction  is  said  to  be  specific;  that  is,  the 
agglutinin  will  agglutinate,  in  general,  only  the  homologous 
organism.  The  term  homologous  is  used  to  indicate  the  relation- 

147 


148  VETERINARY  BACTERIOLOGY 

ship  between  an  antigen  and  its  specific  antibody.  The  serum 
of  a  glandered  animal  is  homologous  for  the  glanders  bacillus,  but 
is  heterologous  for  the  typhoid  bacillus. 

Agglutinins  are  formed  by  the  body  for  most  foreign  cells  which 
may  enter  or  be  injected.  Red  blood-cells,  other  body-cells,  pro- 
tozoa or  bacteria,  may  be  the  antigens  which  provoke  agglutinin 
production.  Under  the  right  conditions  the  clumping  will  occur 
whether  the  cells  be  living  or  dead,  motile  or  non-motile. 

Agglutinogen. — The  antigen  which  causes  the  body  to  react  and 
produce  agglutinins  is  called  an  agglutinogen.  It  is  evident  that 
the  agglutinogen  is  not  the  cell  used  for  injection,  but  some  sub- 
stance produced  by  it.  A  culture  of  Bacillus  typhosus  in  broth 
may  be  passed  through  a  porcelain  filter,  and  the  sterile  filtrate 
will  still  cause  agglutinin  production  when  injected  into  the  animal 
body.  The  agglutinogen  is  either  something  thrown  off  by  the 
antigenic  cell  in  the  process  of  growth,  or  formed  as  a  result  of 
autolytic  disintegration  and  digestion.  Evidently  some  con- 
stituent of  the  cell  excites  the  production  of  the  antibodies  or 
agglutinins,  and  these,  therefore,  unite  with  the  corresponding 
material  in  the  bacterial  or  other  cell. 

Ehrlich's  Theory  of  Agglutinin  Production. — According  to 
Ehrlich,  the  agglutinogen  unites  with  the  receptors  of  the  body- 
cells,  which  are  diverted  in  this  way  from  their  normal  function. 
As  a  result,  there  is  an  overproduction  of  the  receptors  and  they 
are  freed  as  agglutinins.  These  freed  receptors  or  agglutinins 
differ  in  several  ways  from  the  antitoxins,  for  they  not  only  com- 
bine with  the  antigen,  but  they  bring  about  certain  changes  in  it. 
Such  receptors,  to  differentiate  them  from  antitoxins  and  similar 
antibodies  (receptors  of  the  first  order),  are  termed  receptors  of 
the  second  order. 

Constitution  of  the  Agglutinin. — The  agglutinin  may  be  shown 
to  consist  of  two  portions— a  binding  group  and  an  agglutinating 
group.  The  presence  of  the  binding  group,  or  haptophore,  may  be 
shown  by  mixing  bacteria  with  a  serum  containing  the  specific 
agglutinin,  and  centrifuging.  The  supernatant  liquid  will  be 
found  to  have  lost  its  agglutinating  power — that  is,  the  agglutinins 
will  all  have  united  with  the  bacteria  first  added,  and  will  be  removed 
thereby  from  solution.  The  agglutinating  group  of  the  agglutinin 


AGGLUTINATION    AND    PRECIPITATION 


149 


is  called  the  agglutinophore,  the  zymophore,  or  the  zymotoxic  group. 
This  group  is  unstable,  and  may  be  destroyed  by  heating  to  a  tem- 
perature of  60°  to  75°  and  by  acids  and  alkalis.  It  changes  with 
age  slowly.  An  agglutinin  which  has  lost  its  zymotoxic  group  is 
called  an  agglutinoid.  The  agglutinoid  still  fetains  the  capacity 
to  unite  with  the  antigen,  but  has  lost  the  ability  to  act  upon  it. 
The  presence  of  agglutinoids  may  be  demonstrated  by  mixing  a 
serum  containing  them  with  the  homologous  organism,  and  allow- 
ing the  mixture  to  stand  for  a  time.  No  agglutination  will  take 
place,  nor  will  it  occur  when  fresh  agglutinin  is  added.  The 


3.          '  '        '4. 

Fig.  73. — Agglutination  and  formation  of  agglutinins:  1,  Diagrammatic 
representation  of  bacterial  or  other  antigenic  cells  with  fixed  (a)  and  freed  (6) 
agglutinogen  groups.  2,  Union  of  agglutinogen  with  cell  receptors  of  the 
second  order:  a,  Molecule  of  the  cell  protoplasm,  with  a  cell  receptor  of  the  first 
order  (e)  and  of  the  second  order  (6) ,  showing  its  haptophore  (c)  and  its  aggluti- 
nophore or  zymophore  (d);  f,  an  agglutinogen  group  united  to  the  cell  receptor. 
3,  Overproduction  of  cell  receptors  6  and  freed  receptors  or  agglutinin  molecules 
at  c.  4,  Union  of  agglutinin  with  the  bacterial  or  other  cell. 

agglutinoid  unites  with  the  cell  and  blocks  the  union  of  the  ag- 
glutinin. Certain  investigators  have  claimed  to  have  produced 
antiagglutinins  by  the  use  of  agglutinins  as  an  antigen,  inoculating 
them  into  another  species  of  animal.  These  antiagglutinins,  when 
mixed  with  the  agglutinins,  unite  with  them  and  prevent  them  from 
uniting  with  the  homologous  antigen  when  it  is  added. 

Body  or  Somatic  and  Flagellar  Agglutinins. — It  is  probable  that 
agglutinogen  in  motile  organisms  may  originate  in  two  ways — from 
the  flagella  or  from  the  cell-bodies.  Agglutinins  have  been  differ- 
entiated in  such  cases  into  those  that  bring  about  agglutination 


150  VETERINARY   BACTERIOLOGY 

by  combining  with  the  flagella  (flagellar  agglutinins)  and  those 
which  unite  with  the  cell-body  (somatic  or  body  agglutinins). 

Method  of  Agglutinin  Action. — Common  salt  must  be  present 
in  any  serum  which  agglutinates.  Its  function  is  not  thoroughly 
understood,  nor  is  the  general  phenomenon  of  agglutination  itself 
susceptible  of  a  simple  explanation.  The  older  theories  of  the 
change  whereby  the  organisms  are  made  glutinous  by  the  union 
of  the  agglutinin  are  to  be  discarded,  and  the  true  explanation  is 
doubtless  to  be  found  somewhere  in  the  field  of  colloid  chemistry. 
Concerning  the  exact  nature  of  the  change  in  the  cell  we  know 
little  or  nothing.  The  cells  are  certainly  not  seriously  injured; 


\m\mw\\ 


3 

Fig.  74. — Diagrammatic  representation  of  group  agglutination:  Let  1,  2, 
and  3  represent  the  agglutinogen  given  off  by  three  related  species  of  micro- 
organisms. The  organisms  1  and  2  have  agglutinogens  a  and  F  in  common, 
but  in  quite  different  proportions.  No.  3  likewise  has  F  in  common  with  both 
of  the  others,  and  D  in  common  with  1.  If  the  area  covered  in  the  diagrams 
represents  the  relative  proportions  of  agglutinogen  of  each  kind,  the  readiness 
with  which  one  species  may  be  agglutinated  by  a  heterologous  serum  may 
be  understood. 

in  fact,  an  organism  may  grow  luxuriantly  in  a  serum  which  agglu- 
tinates it.  One  of  the  delicate  tests  for  agglutinability  is  to  grow 
the  organism  in  such  a  serum,  and  note  the  production  of  long 
threads  (Pfaundler's  reaction). 

Group  Agglutinins. — The  statement  has  been  made  that  agglu- 
tinins are  specific.  This  must  be  somewhat  modified.  It  has  been 
shown  that  the  serum  homologous  for  a  certain  organism  may 
clump  to  a  less  degree  some  other  species  or  closely  related  forms, 
and  in  rare  instances  forms  quite  unrelated.  This  seems  to  be 
due  to  the  fact  that  not  all  the  agglutinogen  given  off  by  a 
particular  organism  is  of  one  type;  the  agglutinins  produced, 


AGGLUTINATION   AND   PRECIPITATION  151 

therefore,  are  likewise  of  different  types.  It  is  entirely  probable 
that  closely  related  organisms  should  throw  off  some  identical 
agglutinogen,  and,  therefore,  have  some  common  agglutinins. 
Agglutination  of  an  organism  by  a  heterologous  serum  is  termed 
group  agglutination.  The  agglutinins  which  are  specific  for  the 
organism  are  called  its  chief  agglutinins,  and  those  common  to  two 
or  more  organisms  are  termed  coagglutinins.  It  may  sometimes 
be  shown  that  differences  exist  between  the  agglutinins  produced 
by  different  strains  of  the  same  organism.  The  importance  is 
apparent,  therefore,  of  using  care  in  testing  the  agglutinating  power 
of  any  serum  to  dilute  to  such  an  extent  that  the  action  of  the 
coagglutinins  may  be  negligible  and  that  of  the  specific  or  chief 
agglutinins,  recognized. 

Agglutination  Tests  in  Disease  Diagnosis. — The  fact  that  an 
organism  developing  in  the  body  generally  excites  the  production 
of  a  specific  agglutinin  has  led  to  the  wide  use  of  the  fact  in  the 
diagnosis  of  certain  infectious  diseases.  Not  all  diseases  cause 
an  appreciable  production  of  agglutinin.  The  test  is  carried  out 
by  mixing  serum  from  the  suspect  with  the  organism.  If  agglu- 
tination occurs  in  proper  dilution,  it  is  evident  that  the  specific 
organism  is  or  has  been  present  in  the  patient.  The  test  is  fre- 
quently reversed,  and  serum  from  an  animal  showing  high  agglu- 
tinating power  is  used  to  differentiate  between  different  species 
and  races  of  bacteria.  The  principal  disease  organisms  which  cause 
the  production  of  appreciable  amounts  of  agglutinin  are  as  follows : 
Bacillus  typhosus  (typhoid  fever),  Bacillus  paratyphus  (paraty- 
phoid) ,  Bacillus  enteritidis  (meat-poisoning) ,  Bacillus  dysenteric  (ba- 
cillary  dysentery),  Bacillus  coli,  Bacillus  pyocyaneus,  Bacillus  mallei 
(glanders),  Bacillus  pestis  (bubonic  plague),  Bacillus  tuberculosis 
(tuberculosis),  Micrococcus  melitensis  (Malta  fever),  Streptococcus 
pyogenes,  and  some  other  pyogenic  cocci,  Micrococcus  meningitidis 
(epidemic  cerebrospinal  meningitis),  Spirillum  cholera?  (Asiatic 
cholera),  and  others.  The  test  is  not  commonly  used  in  practice 
for  the  recognition  of  all  of  them — some  are  of  experimental  interest 
only. 

The  diagnosis  of  disease  by  agglutination  is  commonly  called 
the  "  Gruber-Widal,"  or  simply  the  "  Widal  "  test.  The  test  may 
be  made  either  by.  observation  of  a  hanging  drop  or  microscopically, 


152 


VETERINARY    BACTERIOLOGY 


or  it  may  be  made  by  naked-eye  observation  of  the  reaction  in 
the  test-tube,  or  macroscopically. 

Microscopic  Widal,  or  Agglutination  Test. — Dilutions  of  the 
serum  are  generally  made  1 : 10,  1 :  20,  and  1 : 40,  or  even  more. 
To  prepare  a  1 : 40  dilution  for  a  hanging  drop,  place  19  loops  of 
physiological  salt  solution,  separately,  upon  a  clean  microscopic 
slide,  then  add  one  loopfulof  serum  to  be  tested,  and  mix  thoroughly 
with  the  diluent.  Place  one  loopful  of  a  suspension  of  the  organ- 
ism (broth  culture  or  suspension  from  the  surface  of  an  agar  slant 
in  physiological  salt  solution)  upon  each  of  two  clean  cover-glasses  ; 
to  one  add  one  loopful  of  the  serum  dilution,  to  the  other  a  loopful 
of  sterile  physiological  salt  solution.  Invert  over  the  cavity  of  a 


A  B 

Fig.  75. — The  Widal  or  agglutination  test  of  the  typhoid  bacillus,  using 
serum  from  a  typhoid  patient:  A,  Check  showing  the  uniform  distribution 
of  the  bacilli;  B,  clumps  of  bacteria  in  the  positive  test  (Jordan). 

hollow-ground  slide  and  examine  microscopically.  The  check 
should  show  the  organisms  uniformly  distributed  over  the  field, 
and  moving  about  actively,  if  the  organism  is  motile.  The  organ- 
isms in  the  other  may  show  no  change,  but  if  the  serum  has 
come  from  a  patient  infected  with  the  organism,  the  bacteria  will 
soon  begin  to  clump  together,  and  in  the  course  of  a  few  minutes 
to  an  hour  practically  all  of  the  organisms  will  be  found  so 
clumped,  very  few  or  none  remaining  isolated  in  the  field.  Motility 
is  lost  in  motile  forms.  The  test  is  a  very  delicate  one,  as  is  evi- 
denced by  the  fact  that  the  agglutination  may  sometimes  be  secured 
in  dilutions  as  great  as  1 :  100,000.  The  higher  the  dilution 


AGGLUTINATION   AND    PRECIPITATION  153 

at  which  agglutination  occurs,  the  greater  is  the  specificity  of 
the  reaction.  As  has  been  seen,  the  dilution  must  be  great  enough 
in  every  case  to  escape  the  activity  of  the  normal  and  the  common 
group  agglutinins.  Serum  from  an  animal  that  has  been  immun- 
ized against  a  specific  organism  may  be  used  in  the  recognition 
of  that  organism.  Typhoid  serum  in  this  way  can  be  used  in  the 
detection  of  the  typhoid  bacillus.  Such  a  test  also  enables  one  to 
differentiate  between  closely  related  forms,  as  the  varieties  of  the 
dysentery  and  of  the  paratyphoid  bacilli. 

Macroscopic  Widal,  or  Agglutination  Test. — A  series  of  small 
test-tubes  is  prepared,  each  tube  containing  a  definite  amount  of  a 
suspension  of  the  organism.  To  these  are  added  varying  quantities 
of  serum,  making  dilutions  of  1 : 10, 1 :  50, 1 : 100, 1 :  200,  and  higher. 
A  positive  reaction  is  indicated  by  the  appearance  of  small  flocculi 
of  bacteria,  which  soon  settle  to  the  bottom,  leaving  the  super- 
natant liquid  clear.  The  reaction  may  not  be  complete  for  several 
hours,  and  the  tubes  should  be  allowed  to  stand  for  twenty-four 
hours  before  making  final  observations.  Check  tubes  should 
always  be  kept  as  controls,  in  which  the  liquid  should  remain 
uniformly  turbid.  Advantage  is  taken  by  certain  manufacturers 
of  the  fact  that  dead  bacteria,  as  well  as  the  living,  may  be 
agglutinated.  They  make  "  agglutinometers  "  containing  all 
the  apparatus  and  materials  for  making  a  complete  test.  The 
bacterial  suspension  supplied  will  keep  for  a  long  period. 

Significance  of  Agglutinins  in  Immunity. — Agglutination  takes 
place  in  the  body  as  well  as  without.  Clumps  of  typhoid  bacilli 
may  be  found  in  various  capillaries  in  cases  of  typhoid.  It  is 
uncertain  what  significance  is  to  be  attached  to  the  phenomenon. 
It  can  scarcely  be  of  advantage,  except  possibly  that  it  prevents 
to  some  degree  the  distribution  of  the  bacteria  through  the  blood. 

Hemagglutinins. — Certain  bacteria,  and  certain  toxic  materials 
from  plants  and  animals,  contain  substances  which  agglutinate 
red  blood-cells.  Such  agglutinins  are  termed  hemagglutinins. 
When  this  agglutination  occurs  in  the  blood,  it  results  in  the 
formation  of  emboli  of  the  red  blood-cells.  These  emboli  are  of 
considerable  significance  in  some  diseases.  Hemagglutinins  may 
also  be  formed  by  the  injection  of  the  red  blood-cells  of  one  species 
into  another. 


154  VETERINARY   BACTERIOLOGY 

Precipitins. — The  precipitins  are  quite  analogous  to  the  ag- 
glutinins.  They  are  formed  as  a  result  of  the  injection  of  a  great 
variety  of  proteins  or  protein  derivatives.  An  antigen  which  in- 
duces the  development  of  precipitins  is  known  as  a  predpitogen. 
The  precipitogen  is  doubtless  the  protein  molecule  itself.  It  con- 
sists essentially  of  a  binding  or  haptophore  group  only.  The 
precipitin  seems  to  be  formed  in  a  manner  similar  to  that  already 
described  for  agglutinins.  It  may  be  shown  to  consist  of  a 
zymophore  or  precipitating  group,  and  a  haptophore  or  binding 
group.  The  zymophore  is  easily  destroyed  by  heat,  but  the  hap- 
tophore is  thermostabile.  A  precipitin  that  has  lost  its  zymophore 
is  known  as  a  predpitoid.  The  precipitins  are  quite  specific,  but 
group  precipitation  will  take  place  when  related  proteins  are 
treated  with  a  serum  homologous  to  one  of  them.  The  blood- 
sera  of  various  ruminants,  for  example,  exhibit  group  precipita- 
tion. An  antiserum  homologous  to  human  serum  will  precipitate 
the  serum  of  anthropoid  apes. 

The  work  of  Nuttall  has  showed  quite  definitely  the  limits 
of  group  agglutination.  He  tested  900  kinds  of  blood,  using  in 
all  30  antisera,  and  made  a  total  of  about  16,000  combinations. 
He  showed  that,  on  the  whole,  the  closer  the  relationship,  the  greater 
the  amount  of  common  or  group  agglutination.  For  example, 
he  determined  that  the  blood  of  apes  of  the  old  world  would  yield 
a  heavier  precipitate  with  human  antiserum  than  would  that  of 
apes  of  the  new  world.  Another  exception  to  specificity  has 
been  found  in  the  protein  of  the  crystalline  lens:  an  antiserum 
for  the  lens  protein  of  man  or  the  ox  will  produce  a  precipitate  in 
solution  containing  the  lens  protein  from  other  animals  not  at  all 
closely  related.  It  has  been  suggested  that  the  cataract  of  the 
eye  may  be  due  to  the  formation  of  an  autoprecipitin  for  the 
protein  of  the  lens  and  its  consequent  partial  coagulation  or  pre- 
cipitation in  situ. 

The  mechanism  of  precipitation  is  not  well  understood,  but 
it  is  probably  to  be  explained  on  the  basis  of  certain  facts  of 
colloid  chemistry.  The  test  is  so  delicate  that  a  positive  reaction 
has  been  secured  with  a  dilution  of  1 :  100,000  of  egg-white,  while 
the  ordinary  protein  tests  of  the  chemist  fail  to  show  1 :  1000. 

Uses  Made  of  the  Precipitation  Phenomenon. — Several  practical 


• 
AGGLUTINATION   AND    PRECIPITATION  155 

applications  have  been  made  of  precipitation  in  the  differentiation 
of  proteins.  These  are  the  recognition  of  blood-stains,  the  differ- 
entiation of  meats  from  different  species  of  animals,  particularly 
horse-flesh  from  beef,  and  the  diagnosis  of  many  other  protein- 
containing  substances,  including  bacteria  and  their  products. 

Recognition  of  Blood-stains. — It  is  sometimes  necessary  in 
murder  trials  to  determine  with  certainty  the  origin  of  a  blood-stain. 
The  fact  that  the  stain  has  been  produced  by  blood  may  be  easily 
demonstrated  by  the  chemist,  but  he  has  no  ready  means  of  telling 
with  certainty  whether  the  blood  is  of  animal  or  human  origin. 
Uhlenhuth  was  the  first  to  call  attention  to  the  value  of  this  test 
in  legal  medicine.  The  precipitation  test,  when  properly  carried 
out,  enables  the  determination  to  be  made  with  a  high  degree  of 
certainty.  An  antiserum  specific  for  human  blood  must  be  se- 
cured first  by  injections  of  human  serum  into  a  rabbit,  at  intervals 
of  a  few  days,  for  a  period  of  several  weeks.  A  bit  of  the  material 
with  the  blood-stain  is  placed  in  a  watch-glass  and  5  c.c.  of  sterile 
physiological  salt  solution  is  added.  This  is  allowed  to  stand  until 
some  of  the  blood  proteins  have  been  dissolved.  This  may  be 
shown  by  blowing  into  the  liquid  through  a  capillary  tube,  when  a 
fairly  permanent  foam  will  be  produced.  If  any  dirt  or  sediment 
appears  in  the  solution,  it  is  removed  by  filtration.  An  effort 
is  made  to  secure  a  dilution  of  the  serum  of  about  1 :  1000.  The 
diluted  serum  is  placed  in  a  series  of  test-tubes,  and  the  specific 
antiserum  is  added.  If  the  blood-stain  is  from  human  blood,  the 
precipitate  will  make  itself  apparent  as  a  clouding  in  the  course  of 
a  few  minutes.  The  reliability  of  the  test  has  been  recognized 
by  the  German  courts,  and  the  results  have  been  accepted  as 
evidence.  By  varying  the  procedure,  the  same  method  may  be 
used  in  differentiating  animal  bloods. 

Differentiation  of  Meats. — Meat  inspection,  particularly  in 
certain  European  countries,  includes  the  differentiation  of  meats. 
In  certain  localities  large  quantities  of  horse-flesh  are  used  for  food, 
but  the  law  forbids  that  horse-flesh  be  sold  as  beef.  There  are  cer- 
tain chemical  differences  between  the  two, — differences  in  the  com- 
position of  the  fat  and  possibly  in  the  abundance  of  glycogen, — but 
these  differences  require  careful  chemical  analysis  and  examination 
for  their  recognition.  The  precipitation  test  furnishes  an  easier 


156  VETERINARY    BACTERIOLOGY 

and  more  reliable  method  for  reaching  the  same  end.  Further- 
more, the  testing  may  be  extended  to  an  examination  of  mixed 
meats,  as  sausages,  and  the  various  kinds  of  meat  present  deter- 
mined. Specific  antisera  must  be  prepared  for  each  of  the  meats 
which  it  is  desired  to  recognize  by  mincing  the  meat  and  soaking 
it  in  physiological  salt  solution,  j  This  material  is  then  used  in  the 
immunization  of  a  rabbit  by  repeated  injections  during  several 
weeks.  The  flesh  to  be  tested  is  likewise  extracted  with  physio- 
logical salt  solution,  the  solution  filtered  and  tested  as  in  the  blood 
diagnosis  with  the  various  specific  antisera.  It  will  give  a  most 
prominent  precipitate  with  its  homologous  antiserum,  and  the 
differentiation  may  thus  be  made. 

Differentiation  of  Bacteria. — It  has  been  found  that  the  injec- 
tion of  the  bacteria-free  filtrates  of  liquid  cultures  of  bacteria  will 
induce  the  production  of  an*  antiserum  specific  for  the  filtrate 
from  that  particular  type  of  organism.  This  method  is  not  as 
reliable  as  the  agglutination  method  of  differentiating  bacteria, 
but  it  may  be  used,  and  is  just  as  specific. 

Similar  tests  have  been  made  to  differentiate  from  each  other 
the  proteins  derived  from  certain  plants  and  plant-seeds.  It  may 
be  stated  that,  in  general,  the  injection  of  any  protein  in  abso- 
lutely pure  condition  will  cause  the  production  of  the  homologous 
specific  antiserum  in  the  animal  injected. 


CHAPTER   XVI 
CYTOLYSINS,  INCLUDING  BACTERIOLYSINS,  AND  HEMOLYSINS 

(Antibodies  of  Ehrlich's  Third  Order) 

THE  use  of  animal,  plant,  or  bacterial  cells  as  antigens  has  been 
found  usually  to  cause  the  development,  by  the  tissues,  of  specific 
antibodies,  which  have  the  power  of  destroying  these  cells,  and 
in  many  cases  of  actually  dissolving  or  digesting  them.  These 
antibodies  are  termed  cytotoxins.  In  most  cases  the  action  is 
lytic  or  dissolving;  the  antibodies  are  cytolysins.  Cytolysins  are 
frequently  subdivided  with  reference  to  their  antigens,  as  bac- 
teriolysins,  hemolysins,  nephrolysins,  etc.  Any  substance  which 
destroys  bacterial  cells  is  said  to  be  bactericidal,  and  this  expression 
is  used  when  the  method  of  cell  destruction  is  not  specified.  Cyto- 
lysins are  produced  by  certain  bacteria,  and  are  also  found  in 
certain  snake  venoms. 

Bacteriolysins  were  first  noted  by  Pfeiffer.  He  found  that 
when  cholera  spirilla  were  injected  into  the  peritoneal  cavity  of  the 
immune  guinea-pig,  the  serous  exudate  rapidly  dissolved  and 
destroyed  them.  This  lytic  action  of  the  blood-serum  is  called 
Pfeiffer's  phenomenon.  Later,  it  was  discovered  that  this  reaction 
would  take  place  just  as  well  in  a  test-tube  (in  vitro)  as  in  the 
animal  body. 

Cytolysins. — Cytolysins  have  been  shown  to  be  made  up  of  two 
elements.  When  a  cytolytic  serum  is  heated  to  56°  for  half  an 
hour,  or  is  allowed  to  stand  for  a  time,  it  loses  its  lytic  property. 
This  is  regained,  however,  when  a  little  fresh  normal  serum  is  added. 
The  normal  serum  is  said  to  reactivate  the  immune  serum.  The 
normal  serum  alone  is  not  lytic,  nor  is  the  immune  serum,  but  when 
mixed,  they  will  destroy  cells.  It  is  evident,  therefore,  that  the 
cytolysin  is  made  up  of  two  constituents,  neither  of  which  can 
act  without  the  other.  The  thermostabile  constituent  of  the  im- 

157 


158  VETERINARY   BACTERIOLOGY 

mime  serum  is  termed  amboceptor;1  the  thermolabile  constituent 
of  normal  serum  is  termed  complement." 

Action  of  Amboceptor. — It  may  be  shown  that  the  amboceptor 
unites  with  the  antigenic  cell  with  which  it  comes  in  contact. 
This  may  be  demonstrated  by  the  following  experiment:  A 
heated  immune  serum  (i.  e.}  one  containing  amboceptor  only) 
is  added  to  the  homologous  cell,  allowed  to  stand  for  a  time,  then, 
by  means  of  repeated  centrifugation  and  washings  with  physiologi- 
cal salt  solution,  the  serum  may  be  completely  removed.  To  the 
washed  cells  normal  fresh  serum  (i.  e.,  containing  complement) 
is  added,  when  cytolysis  may  be  observed.  This  seems  to  show 
that  the  amboceptor  unites  with  the  cell  and  cannot  be  removed 
by  washing,  but  cannot,  on  the  other  hand,  destroy  the  cell  until 
the  complement  is  added. 

Action  of  Complement. — Normal  serum  containing  complement 
only  shows  no  cytolytic  activity.  An  experiment  reciprocal  to 
the  preceding,  the  addition  of  complement  containing  serum  to  a 
suspension  of  cells,  followed  by  centrifugation  and  washing,  and 
the  addition  of  amboceptor,  will  not  result  in  cytolysis.  The 
complement  is  evidently  not  bound  to  the  cell,  and  can  only  be 
attached  through  the  amboceptor.  The  amboceptor  may  be  con- 
sidered as  a  structure  which  links  or  binds  the  complement  to 
the  cell  and  enables  it  to  destroy  such  cell. 

Specificity  of  Amboceptors  and  Complements. — The  amboceptor 
is  formed  usually  as  the  result  of  immunization,  and  is  specific 
for  its  antigen.  The  amboceptor  for  the  red  blood-cells  of  one 
species  of  animal  will  not  unite  with  those  of  an  unrelated  species. 
Inasmuch  as  normal  serum  will  activate  many  different  ambocep- 
tors,  it  has  been  argued  that  all  complement  is  of  a  single  type,  but 
Ehrlich  has  succeeded  in  demonstrating  that  there  are  many 
complements,  and  the  general  activating  power  of  certain  fresh 
sera  for  many  amboceptors  is  due  to  the  multiplicity  of  the  com- 
plements which  they  contain. 

Structure  of  Amboceptor. — There  is  reason  for  believing  that  the 

1  Synonyms  of  amboceptor  are  immune  body,  Imnmnknrpor,  Zwischen- 
korper,  substance    sensibilisatrice,    copula,    desmon,    philocytase,    fixateur, 
preparator,  Hilfkorper; 

2  Synonyms  of  complement  are  addiment,  alexin,  cytase. 


CYTOLYSIXS,    BACTERIOLYSINS,   AXD   HEMOLYSINS  159 

amboceptor  is  made  up  of  two  haptophore  groups,  one  uniting 
with  the  specific  cell,  and  called  the  cytophilous  haptophore,  the 
other  uniting  with  the  complement,  and  called  the  complement- 
ophilous haptophore.  The  injection  of  a  serum  containing  ambo- 
ceptors  into  a  different  species  of  animal  has  been  claimed  to  cause 
the  production  of  anti-amboceptors.  That  these  anti-amboceptors 
are  formed  was  held  because,  by  adding  the  serum  produced  by 
immunization  with  amboceptors  to  a  solution  of  the  amboceptors, 
the  solution  will  be  found  to  have  lost  all  cytolytic  power  when  the 
complement  is  added.  The  fact  that  the  amboceptor  has  the  two 
haptophore  groups  would  make  it  seem  probable  that  two  kinds  of 
anti-amboceptors  may  be  formed,  one  of  which  unites  with  the 
complementophilous,  the  other  with  the  cytophUous,  haptophore. 
That  such  are  actually  present  may  be  demonstrated  by  carefully 
planned  experiments.  Add  the  anti-amboceptor  solution  to  the 
solution  of  amboceptor,  then  add  the  specific  cell  antigen,  wash  the 
cells  repeatedly  with  physiological  salt  solution  by  means  of  centri- 
fugation,  and  add  the  complement.  No  cytolysis  will  take  place, 
but,  on  the  addition  of  fresh  amboceptor,  cytolysis  will  occur. 
It  is  evident  that,  in  the  first  instance,  the  amboceptor  has  been 
prevented  from  uniting  with  the  cell  by  the  cytophilous  anti- 
amboceptor.  The  anti-amboceptor  for  the  complementophilous 
haptophore  may  be  demonstrated  by  adding  amboceptor  to  the 
antigenic  cell;  to  a  portion  add  anti-amboceptor.  To  each  por- 
tion then  add  complement,  and  it  will  be  found  that  no  cytolysis 
occurs  when  anti-amboceptor  has  been  used,  while  it  does  occur  in 
the  other  tube.  The  anti-amboceptor  in  this  case  has  prevented 
the  complement  from  attaching  itself  to  the  amboceptor,  and 
consequently  prevented  cytolysis.  Some  doubt  has  been  thrown 
upon  the  sufficiency  of  the  above  explanation,  for  it  has  been  shown 
that  the  anti-amboceptor  for  goat  anticholera  serum  will  inhibit 
likewise  the  action  of  the  goat  typhoid  serum. 

Structure  of  the  Complement. — The  complement  is  believed  to 
consist  of  two  groups — a  haptophore,  which  unites  with  the  ambo- 
ceptor, and  an  active  or  lytic  group,  the  zymophore.  Careful 
heating  of  complement  is  found  to  destroy  the  zymophore  without 
injuring  the  haptophore.  Such  a  changed  complement  is  called  a 
complementoid.  It  has  been  claimed  that  immunization  of  one 


160 


VETERINARY   BACTERIOLOGY 


species  of  animal  with  the  complement  of  another  results  in  the 
formation  of  anticomplement,  but  more  recent  investigations 
have  thrown  some  doubt  upon  the  sufficiency  of  the  explanation 
offered. 

Ehrlich's  Conception  of  Formation  of  Amboceptor  and  Comple- 
ment.— Ehrlich  calls  an  amboceptor  a  freed  cell  receptor  of  the 
third  order.  Such  a  receptor  he  believes  exists  in  the  form  of  a 
double  haptophore,  and  unites  first  with  food  or  other  materials, 
then,  by  means  of  the  other  haptophore,  with  complement  which 
is  present  in  the  serum.  The  latter  doubtless  normally  brings 


Fig.  76. — Formation  and  action  of  cytolysins:  1,  Bacterial  or  other  cell, 
a,  with  receptors,  b,  which  are  thrown  off  as  antigens,  c;  2,  protoplasmic  mole- 
cule of  the  body  a,  with  a  receptor  of  the  third  order,  6.  This  receptor,  by 
means  of  its  cytophilous  haptophore,  d,  can  unite  with  the  antigen,  e,  and 
by  means  of  its  complementophilous  haptophore,  r,  it  can  unite  with  the 
complement  /.  At  g  is  shown  a  receptor  with  both  haptophores  occupied. 
At  h  is  a  freed  cell  receptor  or  amboceptor;  3,  a  bacterial  or  other  cell,  a,  to  which 
an  amboceptor  has  united  by  means  of  the  receptor  6,  and  with  a  complement 
united  to  c.  This  completes  the  lytic  system,  and  the  cell  may  be  destroyed 
by  the  complement. 


about  changes  of  a  digestive  nature  which  enable  the  cell  proto- 
plasm to  make  use  of  the  food.  The  antigens  used  in  immuniza- 
tion unite  with  such  receptors  and  divert  them  from  their  normal 
function.  Possibly  the  cell  is  injured;  it  is,  at  any  rate,  stimulated 
to  an  overproduction  of  these  receptors,  and  they  are  thrown  free 
in  the  blood  as  amboceptors. 

Group  Cytolysins. — It  may  be  shown  that  related  cells  contain 
some  similar  antigens,  and  that  the  cytolysins  for  one  species  of 
cell  may  dissolve  in  low  dilutions  the  cells  of  another  species. 
This  phenomenon  is  similar  to  that  of  group  agglutination,  and 
seems  to  be  based  upon  the  same  general  facts. 

Bacteriolysins. — Bacteriolysins  are  normally  present  for  certain 


CYTOLYSINS,    BACTERIOLYSINS,    AND   HEMOLYSIXS  161 

bacteria  in  the  blood  of  some  animals,  as  bacteriolysins  for  the 
anthrax  organism  in  dog's  blood.  They  may  be  developed  for 
many  organisms  by  systematic  immunization,  or  they  may  appear 
during  the  course  of  an  infection.  Their  presence  may  be  demon- 
strated in  two  ways — either  by  direct  microscopic  examination  or 
by  plating  methods.  In  the  first  method  the  organisms  are  mixed 
with  the  serum  and  examined  microscopically.  They  can  be 
found,  by  actual  observation,  gradually  to  disintegrate  and  dis- 
appear. The  second  method  offers  certain  advantages.  The 
serum  is  mixed  with  the  bacteria,  and  from  time  to  time  portions 
of  the  mixture  are  plated  out,  and  the  rapidity  of  the  destruction 
determined  by  the  relative  number  of  colonies  that  develop. 
Neisser  and  Wichsberg  have  developed  a  technic  which  enables 
them  to  differentiate  dead  and  living  cells  by  the  fact  that  leuko- 
bases  are  formed  from  methylene-blue  during  life  and  not  after 
death. 

Bacteriolytic  Sera  Used  in  Practice. — By  no  means  all  bacteria 
will  induce  the  production  of  bacteriolysins  in  any  quantity  in 
the  body,  as,  for  example,  the  pyogenic  organisms  and  the  pneu- 
mococcus.  The  members  of  the  intestinal  group  and  the  spirilla 
are  readily  destroyed  thus.  Antisera  have  been  prepared  and 
used  for  several  of  the  latter.  In  the  manufacture  of  the  antisera, 
either  living  or  dead  organisms  may  be  used.  Methods  of  titra- 
tion,  whereby  the  actual  bacteriolytic  content  of  the  serum  used 
may  be  known,  have  not  thus  far  been  developed.  The  antisera 
generally  have  other  antibodies  developed  in  addition  to  the  bac- 
teriolysins. Plague  serum  and  that  of  swine  erysipelas  contain 
some  bacteriolysins,  and  probably  the  same  is  true  of  the  sera  used 
in  immunizing  against  hog-cholera  and  against  rinderpest. 

Bacteriolytic  sera  for  passive  immunization  are  prepared  by  the 
methodical  introduction  of  the  organism  into  the  animal  body. 
Either  the  first  injections  must  be  made  with  dead  organisms  or 
an  animal  which  has  recovered  from  an  attack  of  the  disease 
must  be  chosen.  The  animal  is  hyperimmunized  by  increas- 
ing doses  of  the  virulent  organism  after  the  first  establishment  of 
immunity.  This  results  in  the  accumulation  of  considerable 
quantities  of  immune  substances  (amboceptors)  in  the  blood. 
This  serum  may  then  be  used  in  passive  immunization.  Vaccina- 
11 


162  VETERINARY   BACTERIOLOGY 

tion,  or  the  injection  of  dead  or  living  bacteria  into  the  body,  with 
the  resultant  development  of  an  active  immunity,  owes  its  effi- 
ciency, in  some  cases  at  least,  to  the  production,  by  the  body,  of  the 
bacteriolysins  specific  for  the  organism. 

Bacteriolytic  sera  for  passive  immunization  are  not  employed 
against  many  diseases.  Some  organisms,  as  has  been  stated,  cause 
the  production  of  little  or  no  bacteriolysin.  The  bacteriolysin 
developed  in  the  blood  of  one  species  is  not  always  suitable  for  the 
passive  immunization  of  another.  There  "have  been  various 
reasons  advanced  for  this  fact:  the  injection  of  the  foreign  serum 
may  cause  the  production  of  anti-amboceptors,  or  of  anticom- 
plements,  or  of  some  other  antibody  which  would  inhibit  the  lytic 
action  of  the  injected  serum.  Complement  soon  disappears  from 
an  immune  serum;  therefore  the  amboceptors  are  dependent  for 
their  activation  upon  the  normal  complement  of  the  blood.  This 
complement  may  not  in  all  cases  be  suitable  for  combination  with 
the  particular  amboceptor  used,  and  the  lytic  activity  be  thus 
inhibited.  When  the  antiserum  to  be  used  for  passive  immuni- 
zation comes  from  the  same  species  of  animal,  as  is  the  case  in 
immunization  against  hog-cholera  and  rinderpest,  these  objections 
do  not  seem  to  obtain. 

Hemolysins. — Hemolysins  for  the  red  blood-cells  of  one  animal 
usually  may  be  obtained  by  injection  of  these  into  another. 
They  are  divided  into  three  types,  the  classification  being  made 
upon  the  basis  of  relationship.  Heterolysins  are  developed  by  the 
injection  of  the  red  blood-cells  of  one  species  into  another;  isolysins 
by  the  red  blood-cells  of  one  animal  into  another  animal  of  the  same 
species,  and  autolysins  by  an  individual  for  his  own  red  blood-cells. 
The  last  two,  particularly  the  autolysins,  are  not  easily  produced 
or  demonstrated. 

The  study  of  hemolysis  has  proved  of  great  value  in  two  ways: 
First,  hemolysis  is  a  phenomenon  that  may  be  easily  observed, 
and  it  has  been  used,  therefore,  more  than  any  other,  in  the  study 
of  the  nature  and  activity  of  cytolysins.  Hemolysis  is  readily 
detected,  because  the  hemoglobin  escapes  into  the  solution  and  it 
remains  permanently  red,  while  unhemolyzed  red  blood-corpuscles 
soon  sink  to  the  bottom,  leaving  the  blood-serum  clear  and  color- 
less. Second,  they  are  made  of  indirect  use  in  the  diagnosis  of 


I 

CYTOLYSINS,   BACTERIOLYSINS,    AND   HEMOLYSINS  163 

certain  diseases,  and  in  the  recognition  of  certain  organic  sub- 
stances. This  second  use  is  called  variously  absorption  or  fixation 
of  complement,  or,  after  the  name  of  its  discoverers,  the  Bordet- 
Gengou  phenomenon. 

Fixation  of  Complement  and  Its  Utilization. — The  method  of 
complement  fixation  is  one  that  enables  us  to  determine  the  pres- 
ence or  absence  of  cytotoxic,  cytolytic,  or  similar  antibodies  in  a 
serum.  Inasmuch  as  such  substances  are  generally  present  in  the 
serum  of  a  diseased  individual,  the  determination  of  their  presence 
may  constitute  in  such  cases  a  diagnosis  of  the  disease.  A  specific 
example,  the  demonstration  of  fixation  of  complement  by  use  of 
antityphoid  serum,  will  first  be  discussed.  Five  different  solutions 
are  always  needed  in  carrying  out  a  test  of  this  kind. 

1.  Suspension  of  Bacillus  typhosus  (antigen). 

2.  Heated  serum  of  rabbit  immunized  against  1  (amboceptor). 

3.  Normal  serum,  usually  taken  from  guinea-pig  (complement). 

4.  Red  blood-cells.     Those  of  sheep  generally  used  (antigen). 

5.  Heated  serum  of  rabbit  immunized  against  4  (amboceptor). 
Suppose  that  1  and  2  are  mixed,  and  a  small  amount  of  3  added. 

Evidently  the  complete  bacteriolytic  system  is  present  and  bac- 
teriolysis should  occur.  This  is  difficult  to  demonstrate  micro- 
scopically, however,  and  would  not  be  demonstrated  at  all  in  that 
manner  if  an  excess  of  the  first  antigen  or  suspension  of  bacilli  is 
used.  It  is  evident  that  the  complement  added  will  be  used  up  or 
"  bound  "  by  the  combination  of  bacillus  and  amboceptor.  No.  4 
and  o  are  next  added.  They  unite  with  each  other,  but,  as  all 
the  complement  has  been  used  up,  there  can  be  no  hemolysis. 
The  fact  that  hemolysis  does  not  occur  is  proof,  therefore,  that  the 
complement  has  been  removed.  Suppose  that,  instead  of  the  blood 
of  a  rabbit  that  has  been  intentionally  immunized  to  the  disease, 
the  serum  from  a  patient  suspected  of  having  the  disease  is  used. 
If  hemolysis  does  not  occur,  then  amboceptors  for  B.  typhosus 
are  present  in  the  patient's  blood,  and  the  diagnosis  of  the  disease 
would  be  positive.  If  hemolysis  does  occur,  evidently  the  patient's 
serum  lacks  amboceptor  specific  for  typhoid,  and  the  diagnosis 
would  be  negative.  The  test  may  be  reversed  also,  and  some 
organism  suspected  of  being  Bacillus  typhosus  may  be  substituted 
for  No.  1,  and  the  same  technic  carried  through.  A  lack  of  hemo- 


164  VETERINARY   BACTERIOLOGY 

lysis  would  indicate  that  the  organism  had  bound  the  amboceptor 
and  complement,  and,  therefore,  was  Bacillus  typhosus  in  fact, 
while  hemolysis  would  indicate  the  reverse.  The  accompanying 
figure  will  make  this  clearer.  In  carrying  out  a  test  of  this 
kind  it  is  necessary  to  arrange  a  very  complete  series  of  checks. 
When  properly  managed,  the  method  is  very  accurate  and  has 
yielded  results  of  value  in  many  cases. 


C. 

Fig.  77. — Fixation  or  absorption  of  complement:  A,  Diagrammatic  rep- 
resentation of — 1,  Bacterial  cell,  a  (B.  typhosus  in  text),  with  antigen  b;  2, 
amboceptor  for  the  antigen  of  (1)  (the  antityphoid  serum);  3,  normal  com- 
plement of  the  guinea-pig's  blood  with  the  haptophore  c  and  the  zymophore  6. 
4,  red  blood-cell  (sheep)  with  the  antigenic  receptors  6;  5,  amboceptor  from  the 
blood  of  the  rabbit  immunized  against  4.  B,  1,  Bacterial  cell  -f  amboceptor 
+  complement  makes  a  complete  lytic  series;  bacteriolysis  occurs,  and  the 
complement  is  removed  from  solution;  2,  the  red  blood-cell  +  its  amboceptor; 
no  hemolysis,  as  the  complement  has  been  used  by  the  other  series.  C,  1, 
Bacterial  cell  not  B.  typhosus,  to  which  amboceptor  (2)  cannot  unite,  hence  the 
complement  is  not  removed  from  the  serum,  and  when  the  red  blood-cells 
and  their  specific  amboceptor  are  added,  a  complete  hemolytic  system  (3)  is 
formed  and  hemolysis  occurs. 

This  test  has  been  most  used  in  the  diagnosis  of  syphilis,  the 
antigen  being  secured  from  a  macerated  syphilitic  fetus  and  the 
amboceptor  from  the  blood  of  the  suspect.  This  is  known  as  the 
Wassermann  syphilis  test.  A  similar  test  may  be  used  for  diag- 
nosis in  other  diseases.  The  same  method  may  be  used  also  in 
the  differentiation  of  proteins.  In  this  case  the  antigen  used  is  the 
protein,  and  the  first  amboceptor  is  in  the  serum  of  an  animal 
immunized  against  this  protein.  The  method  is  even  more  reliable 
than  the  precipitation  tests  already  discussed,  but  is  more  difficult 


CYTOLYSINS,    BACTERIOLYSINS,   AND   HEMOLYSINS  165 

to  carry  out,  hence  is  less  frequently  used.     It  may  be  used  for  the 
identification  of  blood-stains. 

Cytotoxins. — Antisera  have  been  prepared  for  a  number  of 
different  body-cells,  as  epithelial  cells,  spermatozoa,  and  leuko- 
cytes. Within  limits  they  seem  to  be  specific.  It  is  believed 
that  some  pathological  conditions  in  the  body  are  produced  by 
autocytotoxins,  substances  produced  by  the  body  poisonous  for  its 
own  tissues. 


CHAPTER  XVII 

OPSONINS  AND   PHAGOCYTOSIS 

IT  was  observed  by  Parum  as  early  as  1874  that  decay-producing 
bacteria  quickly  disappeared  when  introduced  into  the  body,  and 
could  not  be  demonstrated  in  the  blood  or  other  tissues.  To 
Metchnikoff,  however,  must  be  given  the  credit  of  elaborating  the 
theory  of  phagocytosis.  By  the  term  phagocytes  (Gr.  phagein,  to 
eat)  is  meant  any  body-cell  which  is  capable  of  taking  up  and 
destroying  other  cells,  usually  those  of  foreign  origin.  These 
phagocytes  are  in  some  cases  fixed  body-cells,  but,  for  the  most  part, 
are  leukocytes  or  white  blood-cells  of  different  kinds.  Upon  the 
phagocytic  activity  of  the  body-cells  Metchnikoff  established  his 
theory  of  immunity.  It  may  be  stated  briefly  as  follows:  If  an 
animal  is  either  naturally  or  artificially  immune  to  a  disease,  it 
means  that  the  invasion  of  the  body  by  organisms  is  followed  by  a 
struggle  between  the  organisms  and  the  phagocytes.  These 
phagocytes  ingest  the  organisms  and  render  them  harmless. 
Metchnikoff  subdivides  the  phagocytes  in  two  ways — first,  on  the 
basis  of  their  morphological  and  functional  behavior,  and,  second, 
on  the  basis  of  their  relationship  to  the  surrounding  tissue,  i.  e., 
some  are  mobile,  others  are  fixed.  The  leukocytes  in  the  blood  are 
in  part  the  free-moving  phagocytes.  The  small  lymphocytes  are 
not  known  to  have  any  phagocytic  power,  and  it  is  probable  they 
are  not  endowed  with  active  motion.  Most  cells  have  not  been 
shown  to  be  phagocytic.  The  polymorphonuclear  and  the  large 
mononuclear  leukocytes  are  the  phagocytes  par  excellence.  Certain 
fixed  cells  of  the  lymph-nodes  and  the  spleen  are  likewise  active 
phagocytes. 

Metchnikoff  terms  the  polymorphonuclear  leukocytes  the 
microphages  (Gr.  micros,  small;  phagein,  to  eat),  and  the  large 
mononuclear,  the  macrophages  (Gr.  macros,  large;  phagein,  to  eat), 


OPSONINS  AND   PHAGOCYTOSIS  167 

and  believes  that  they  perform  somewhat  different  functions  in  the 
body. 

Wright  and  Douglas,  in  1902,  published  observations  that 
threw  much  needed  light  on  the  theory  of  phagocytosis.  They 
showed  that,  in  most  cases,  at  least,  the  bacteria  would  not  be 
destroyed  by  the  white  blood-cells  or  phagocytes  in  the  absence 
of  blood-serum.  They  showed  that  the  serum  contained  something 
that  rendered  the  bacteria  positively  chemotactic  or  attractive 
for  the  phagocytes,  and  they  accordingly  named  this  something 
opsonin  (Gr.  opsonein,  to  prepare  a  meal). 

Opsonins. — Opsonins  may  be  shown  to  be  present  in  certain 
sera  by  the  following  experiment:  Blood  of  a  suitable  animal  is 
drawn  into  citrate  solution  and  centrifuged,  thus  throwing  the 


H,  Si 

Fig.  78. — Phagocytosis  by  human  leukocytes:  1,  Micrococcus  aureus;  2, 
Bacillus  dysenteries;  3,  B.  typhosus;  4,  B.  tuberculosis;  5,  Micrococcus  meningi- 
tidis  (1,  2,  and  3  adapted  from  Levaditi  and  Inman,  4  from  Freeman,  and 
5  from  Councilman). 


cellular  elements  to  the  bottom.  The  top  layer  of  corpuscles, 
or  "  cream/'  containing  a  large  proportion  of  leukocytes,  is  pipetted 
off,  and  mixed  with  physiological  salt  solution  and  again  centflfuged. 
A  second  washing  and  centrifugation  removes  all  traces  of  adher- 
ent serum.  The  corpuscles  are  then  mixed  with  a  suspension  of 
the  bacteria  and  incubated  for  fifteen  minutes.  At  the  end  of 
that  period  mounts  are  prepared,  stained  with  a  suitable  blood- 
stain, such  as  Jenner's  or  Wright's,  and  examined.  The  bacteria 
will  not,  in  general,  be  engulfed  by  the  phagocytes.  A  similar 
series  carried  through,  but  with  the  addition  of  a  little  immune 
serum,  would  exhibit  marked  phagocytosis,  and  considerable 
numbers  of  the  microorganisms  would  be  found  within  the  leuko- 


168  VETERINARY   BACTERIOLOGY 

cytes.  Evidently  the  presence  of  the  serum,  or  rather  the  op- 
sonin  which  it  contains,  induces  phagocytosis.  Opsonins  are 
believed  so  to  change  or  alter  the  bacteria  as  to  make  them  posi- 
tively chemotactic  for  the  phagocytes.  This  does  not  mean  that 
the  bacteria  are  destroyed,  for  there  is  no  evidence  that  they  are 
in  any  way  injured.  That  it  is  an  alteration  of  the  organisms, 
and  not  a  mere  stimulation  of  the  phagocytic  cell,  may  be  shown  as 
follows:  A  suspension  of  washed  corpuscles  prepared  as  before 
is  treated  with  immune  serum,  allowed  to  stand,  and  then  washed. 
When  mixed  with  the  bacterial  suspension,  no  phagocytosis  occurs; 
evidently  the  opsonin  is  not  bound  to  the  phagocytes,  nor  does  it 
stimulate  them  when  they  are  brought  in  contact  with  the  bacte- 
rial cells.  The  converse  of  this  experiment  may  be  tried,  the  bac- 
teria added  to  the  immune  serum,  and  then  centrifuged  out  and 
washed  free  from  all  the  serum  with  salt  solution.  When  these 
organisms  are  added  to  a  suspension  of  the  washed  leukocytes, 
active  phagocytosis  occurs.  The  opsonin  is  bound  to  the  bacterial 
cell,  and  makes  it  in  a  sense  attractive  to  the  leukocytes.  The 
negative  chemotaxis  is  converted  by  this  union  into  a  positive 
chemotaxis.  The  action  of  the  opsonin  has  been  likened  to  that 
of  an  amboceptor,  for  it  links  up  the  bacteria  and  the  white  blood- 
cells.  The  opsonins  are,  however,  not  identical  with  bacteriolytic 
amboceptors. 

Opsonins  for  some  organisms  are  quite  constantly  present  in 
the  blood.  For  example,  human  blood  contains  opsonins  for  the 
organisms  producing  bubonic  plague,  Malta  fever,  pneumonia, 
dysentery,  Asiatic  cholera,  and  for  the  pyogenic  organisms. 
These  are  called  normal  opsonins.  Immune  opsonins  are  those 
produced  as  a  result  of  infection  or  immunization.  It  is  also 
probable  that  some  species  of  bacteria  may  be  taken  up  by  phago- 
cytes in  total  absence  of  opsonins.  On  the  other  hand,  it  is  be- 
lieved that  certain  bacteria,  when  they  invade  the  body,  may 
develop  a  resistance  to  phagocytosis.  The  appearance  of  the 
capsule  about  the  anthrax  bacillus  in  the  blood  has  been  thought 
to  be  a  protective  agency  of  this  character.  Whether  the  im- 
mune and  the  normal  opsonins  are  identical  is  a  moot  question. 
Probably  they  may  differ,  it  being  contended  that  the  normal 
opsonin  is  simply  the  normal  complement  of  the  serum,  for  it  has 


OPSOXIXS   AND    PHAGOCYTOSIS  169 

been  found  to  be  thermolabile  (58°  to  60°  destroys).  The  immune 
or  specific  opsonin  is,  on  the  other  hand,  relatively  thermostabile. 
Opsonic  Index. — It  is  sometimes  advisable  to  compare  the 
opsonic  content  of  the  serum  of  a  diseased  animal  with  that  of  a 
healthy  individual.  The  ratio  between  the  two  sera  may  be 
determined  by  the  relative  effect  they  have  on  the  rapidity  of 
phagocytic  action.  It  is  customary,  though  not  in  all  cases 
necessary,  to  use  the  leukocytes  of  the  species  of  animal  under 
investigation.  The  technic  of  the  operation  is  as  follows: 


Fig.  79. — Standardization  of  bacterial  emulsion.     A  photomicrograph  showing 
the  bacteria  and  the  red  blood-cells  mixed  (Miller). 

Preparation  of  Leukocytes. — These  may  be  secured  by  bleeding 
into  citrate  solution  (1  per  cent,  in  physiological  salt  solution)  and 
centrif  uging.  Pipette  off  the  serum  and  citrate  solution,  add  physio- 
logical salt  solution,  mix,  and  again  centrifuge ;  repeat  the  washing 
and  centrifuging  to  remove  the  last  traces  of  serum.  Carefully 
pipette  off  the  upper  layer  of  corpuscles,  rich  in  leukocytes,  and 
keep  in  a  small  test-tube.  Sometimes  the  defibrinated  whole  blood 
is  used.  In  other  cases,  particularly  with  small  experimental 
animals,  an  intraperitoneal  injection  of  bouillon  is  made,  and  the 
leukocyte-rich  exudate  removed  from  the  peritoneal  cavity  in  the 
course  of  a  few  hours. 


170 


VETERINARY  BACTERIOLOGY 


Preparation 
present    in   a 


of  Bacterial  Emulsion. — The  organisms  must  be 
perfectly  homogeneous  suspension.  With  most 
organisms  a  twenty-four-hour  slant  agar 
culture  may  be  used,  the  growth  scraped 
off  into  sterile  physiological  salt  solution, 
triturated,  and  diluted  until  it  shows  only 
a  faint  opalescence.  Filtration  is  sometimes 
necessary.  This  technic  must  be  varied 
somewhat  for  different  organisms. 

Preparation  of  Serum. — Both  normal 
serum  and  serum  from  the  animal  to  be 
tested  are  secured  by  bleeding,  allowing 
the  blood  to  clot,  centrifuging,  and  taking 
off  the  serum  with  a  pipette  and  transferring 
to  a  small  tube.  Before  use,  the  serum  is 
sometimes  diluted — usually  ten  times. 


• 

± 


Fig.  80. — Opsonizing 
pipette  containing  the 
leukocytes,  bacteria, 
and  serum  (Miller).  Fig.  81. — Opsonic  incubator  (Miller). 

Technic  of  Test. — A  capillary  pipette  with  a  fine  bore  is  prepared 
from  glass  tubing,  and  a  mark  is  made  a  short  distance,  usually 


OPSONINS  AND   PHAGOCYTOSIS  171 

about  2  cm.,  from  the  end.  The  suspension  of  leukocytes  is  then 
drawn  up  to  the  mark,  then  an  air-bubble  is  admitted,  then  the 
serum  to  be  tested  to  the  same  mark,  a  second  air-bubble,  and 
finally  an  equal  amount  of  the  bacterial  suspension.  The  contents 
of  the  tube  are  blown  out  upon  a  glass  plate  or  into  a  watch-glass 
and  thoroughly  mixed.  This  mixture  is  again  drawn  some  dis- 
tance into  the  capillary  pipette  and  the  end  sealed  in  the  flame. 
It  is  then  kept  at  37°  for  fifteen  minutes,  preferably  in  an  opsonic 
incubator,  where  it  may  be  rotated  frequently  to  secure  thorough 
mixture.  The  end  is  then  broken  from  the  pipette,  and  smears 
are  made  and  stained  with  some  good  blood-stain,  such  as  Wright's 
or  Jenner's  modification  of  the  Romanowsky,  which  will  stain 
bacteria.  Special  staining  methods  must  sometimes  be  used,  as  in 
the  examination  of  the  tubercle  bacillus. 

Determination  of  the  Opsonic  Index. — The  total  number  of 
bacteria  ingested  by  100  polymorphonuclear  leukocytes  is 
counted.  This  number  is  determined  both  with  normal  serum 
and  the  serum  to  be  tested.  The  ratio  between  the  number  of 
organisms  ingested  by  the  leukocytes,  when  treated  with  the 
specific  serum  and  when  treated  with  normal  serum,  is  called  the 

opsonic  index.  Let  y  equal  opsonic  index  where  a  is  average 
number  of  bacteria  ingested  per  leukocyte  when  treated  with  a 
specific  serum;  b  is  average  number  of  bacteria  ingested  per  leuko- 
cyte with  normal  serum.  In  general,  the  opsonic  index  is  found  to 
be  low  (less  than  unity)  in  chronic  bacterial  infections. 

McCampbeWs  Modification  of  Opsonic  Test. — McCampbell  has 
considerably  shortened  the  technic  in  veterinary  practice  as  fol- 
lows: The  bacterial  suspension  containing  0.8  per  cent,  sodium 
citrate  is  drawn  to  the  0.5  mark  on  the  leukocyte  pipette  of  a 
hemocytometer,  the  specific  blood  to  be  tested  is  drawn  to  the 
same  mark,  then  all  drawn  into  the  bulb,  mixed,  replaced  in  the 
capillary  tube,  and  the  whole  wrapped  with  a  rubber  band  and 
incubated.  The  same  procedure  is  carried  through  with  normal 
blood.  The  disadvantages  of  this  method  are  the  presence  of 
sodium  citrate,  which  interferes  to  a  slight  degree  with  action  of 
opsonins,  and  the  use  of  two  lots  of  leukocytes  from  two  animals, 
one  diseased  and  the  other  normal.  Smears  and  examinations 


172  VETERINARY   BACTERIOLOGY 

are  made  as  in  the  preceding.  It  is  possible  there  may  sometimes 
be  intrinsic  differences  in  these  leukocytes,  which  render  direct 
comparison  between  their  activity  somewhat  misleading.  These 
difficulties  are  more  than  outweighed  by  the  increased  ease  of 
manipulation,  and  good  results  are  commonly  secured. 

The  variation  in  the  opsonic  content  of  a  patient's  blood  gives 
valuable  information  relative  to  the  development  of  resistance. 
As  stated  above,  in  many  chronic  infections  the  opsonic  index 
is  below  unity.  In  the  treatment  of  tuberculosis  in  the  human, 
by  means  of  minute  injections  of  tuberculin,  the  determination 
of  the  opsonic  index  has  given  much  information  of  practical 
nature.  The  injection  of  bacterial  vaccines  or  bacterins  which 
have  been  carefully  studied  is  followed  at  first  by  an  initial  lower- 
ing of  the  index.  This  is  called  the  negative  phase.  Later,  the 
index  should  rise  above  unity,  when  the  positive  phase  is  said  to  be 
established.  If  the  index  can  be  kept  above  unity,  the  prognosis 
is  generally  regarded  as  favorable. 

Methods  of  Opsonic  Immunization. — Immunization  due  to 
increased  opsonin  is  generally  brought  about  by  the  introduction  of 
bacteria  or  their  products  into  the  body.  Such  an  immunity  is, 
therefore,  active.  There  is  good  reason  to  believe  that  injection 
of  an  opsonic  serum  in  some  cases  confers  some  passive  immunity. 
The  most  commonly  practised  method  of  increasing  the  opsonin 
content  of  the  blood  is  by  vaccination.  This  may  be  defined  as 
the  injection  or  inoculation  with  organisms  or  their  products  in 
such  a  way  that  the  disease  produced  will  run  a  benign  course. 
Such  a  procedure  in  many  cases  induces  the  production  of  bac- 
teriolysins,  in  others  opsonins,  and  in  still  others  both.  Vaccines 
may  consist  of  either  living  or  dead  organisms.  If  the  former, 
they  may  be  either  virulent  or  attenuated.  Vaccination  with  viru- 
lent organisms  is  not  frequently  practised.  Some  organisms  do 
not  produce  typical  and  serious  diseases  unless  they  enter  the 
body  by  a  certain  channel.  The  injection  of  the  Asiatic  cholera 
organism  subcutaneously  is  not  followed  by  any  serious  results, 
but,  when  the  alimentary  tract  is  the  infection  atrium,  the  char- 
acteristic symptoms  of  the  disease  are  produced.  This  fact  has 
been  used  as  the  basis  for  practical  vaccination  against  this  dis- 
ease. Usually  vaccines  consist  of  attenuated  organisms.  Atten- 


OPSONINS   AND    PHAGOCYTOSIS  173 

uation  or  weakening  or  decrease  of  virulence  is  accomplished  in 
several  ways:  in  some  cases  by  growing  at  high  temperatures,  as 
for  anthrax,  heating  to  temperatures  just  below  the  thermal 
death-point,  as  in  blackleg,  by  animal  passage,  as  in  small-pox,  or 
by  growth  on  artificial  media,  as  with  members  of  the  hemorrhagic 
septicemia  group. 

Vaccines  consisting  of  dead  organisms  are  called  bacterins. 
These  are  prepared  and  sold  commercially  under  various  trade 
names.  They  usually  consist  of  pyogenic  organisms  which  have 
been  killed  by  heat.  The  injection  of  these  bacterins  has  been 
shown  to  increase  the  opsonic  index  for  specific  organisms  when 
properly  administered.  It  has  been  found,  however,  that  there  are 
many  strains  of  some  of  the  pyogenic  bacterial  species,  for  example, 
of  Streptococcus  pyogenes,  and  that  vaccination  against  one  strain 
does  not  always  protect  against  another.  Vaccines  are,  therefore, 
prepared  containing  organisms  isolated  from  as  many  sources 
as  possible  and  containing  many  races  or  strains.  Such  a  vaccine 
is  called  polyvalent,  a  vaccine  containing  but  one  strain,  univa- 
lent. 

Autogenic  Vaccines. — Inasmuch  as  it  cannot  be  readily  deter- 
mined to  what  strain  a  particular  organism  causing  a  chronic 
bacterial  infection,  such  as  suppuration  in  a  fistula,  belongs,  it  is 
sometimes  desirable  to  prepare  a  vaccine  of  this  particular  organ- 
ism for  this  single  case.  A  vaccine  prepared  in  this  manner,  to  be 
used  in  the  animal  from  which  the  organism  was  isolated,  is  said 
to  be  autogenic.  The  use  of  autogenic  vaccines,  carefully  prepared 
and  standardized,  has  proved  successful  in  many  cases  in  the  treat- 
ment of  stubborn  and  chronic  suppurative  conditions.  The  tech- 
nic  of  preparation  and  use  follows. 

Isolation  of  Organism. — In  many  instances  a  slant  agar  culture, 
made  directly  by  securing  material  on  a  sterile  platinum  loop 
from  the  deeper  parts  of  the  wound  or  abscess,  will  prove  to  contain 
only  the  causal  organism.  In  most  cases,  however,  it  will  be  found 
advisable  to  plate  directly  in  agar  and  incubate  at  blood-heat. 
An  examination  of  the  colonies  will  reveal  the  causal  organism 
(or  organisms  in  a  mixed  infection). 

Preparation  of  Vaccine  (McCampbell). — Into  each  of  two  flat 
flasks  or  bottles,  100  c.c.  of  nutrient  agar  is  placed,  sterilized,  and 


174  VETERINARY   BACTERIOLOGY 

slanted.  Streaks  of  the  organism  are  made  the  full  length  of  the 
slant  and  incubated  twenty-four  hours  at  blood-heat.  The  water 
of  condensation  is  removed  without  disturbing  the  growth,  and 
100  c.c.  of  sterile  physiological  salt  solution  added.  The  growth 
is  carefully  scraped  from  the  surface  with  a  long  sterile  glass  rod; 
the  suspension  is  placed  in  a  sterile  flask  and  shaken  thoroughly. 

Standardization  of  the  Vaccine. — Before  injections  are  made, 
the  number  of  bacteria  present  per  given  volume  of  the  suspension 
must  be  known  in  order  that  accurate  dosage  may  be  determined. 
The  standardization  may  be  effected  by  direct  count  or  by  use  of 
the  nephelometer.  In  the  first  method,  equal  parts  of  the  bac- 
terial suspension  and  of  defibrinated  human  blood  are  thoroughly 
mixed,  a  smear  prepared,  and  stained  with  a  blood-stain  or  carbol- 
thionin.  The  number  of  red  blood-cells  on  several  fields  and  the 
number  of  bacteria  on  these  fields  are  counted  (see  Fig.  79) .  The 
number  of  red  blood-cells  may  be  taken  as  5,000,000  per  cubic 
millimeter.  The  number  of  bacteria  per  cubic  millimeter  will  be 
found  by  the  following  proportion: 

5,000,000  :  x  : :  number  red  blood-cells  per  field  :  number  bacteria  per  field. 

Suppose  20  red  blood-cells  per  field,  and  50  bacteria,  the  ratio 
becomes — 

5,000,000  :  x  ::  20  :  50  or  x  (number  of  bacteria  per  cubic  centimeter)  = 

•  12,500,000. 

To  convert  the  number  of  bacteria  per  cubic  centimeter,  multiply 
by  1000,  which  in  this  case  would  give  12,500,000,000.  It  is  cus- 
tomary to  dilute  the  bacterial  suspension  so  that  each  cubic  cen- 
timeter will  contain  a  number  of  bacteria  that  is  readily  reckoned, 
usually  50,000,000  per  cubic  centimeter. 

The  nephelometer  is  an  instrument  described  by  McFarland, 
in  which  tubes  containing  varying  amounts  of  barium  sulphate 
in  suspension  in  water  are  observed  side  by  side  with  tubes  of  equal. 
size  containing  the  bacteria.  After  the  tubes  containing  barium 
sulphate  have  been  compared  as  to  opacity  with  tubes  containing 
known  numbers  of  bacteria  per  cubic  centimeter,  they  may  be 
used  in  determining  the  number  present  in  other  bacterial  suspen- 
sions. 


OPSONINS  AND   PHAGOCYTOSIS  175 

Passive  Opsonic  Immunization. — It  is  probable  that  some  anti- 
bacterial sera,  as  the  antimeningococcic  serum,  owe  in  part  their 
immunizing  and  their  curative  powers  to  the  presence  of  opsonins. 
The  part  that  these  opsonins  may  play  in  passive  immunization 
is  not  thoroughly  understood. 


CHAPTER  XVIII 

ANAPHYLAXIS  AND  HYPERSUSCEPTIBILITY 

THE  body  normally  takes  its  food  through  the  intestinal  tract, 
or  by  enteral  introduction.  Any  foreign  proteins  introduced  in 
this  manner  are  generally  changed  by  digestion,  so  that  they  may 
be  used  by  the  body-cells  or  assimilated.  When  introduced  by 
par  enteral  injection  (outside  the  alimentary  canal,  as  subcu- 
taneously,  intravenously,  etc.)  antibodies  of  different  kinds  are 
produced.  Thus  far  we  have  considered  three  antibodies  which 
antagonize  bacteria  or  other  antigens  which  are  introduced,  but 
there  exists  still  another  type  of  body  reaction,  sensitization  toward, 
rather  than  immunization  against,  an  antigen.  This  phenomenon 
is  exhibited  only  under  certain  conditions,  and  the  body  is  then 
said  to  show  anaphylaxis  or  hyper  sensitiveness  to  the  antigen. 
The  significance  can  best  be  understood  by  a  study  of  specific 
examples.  A  number  of  these  have  been  noted  independently  and 
are  deserving  of  mention. 

Phenomenon  of  Arthus. — Arthus  injected  rabbits  subcu- 
taneously  with  horse  serum  at  six-day  intervals.  The  first  three 
injections  were  readily  absorbed  by  the  tissues,  the  fourth  was 
followed  by  some  edema  at  the  site  of  injection,  and,  after  the 
sixth  or  seventh  injection,  the  skin  at  the  site  became  gangrenous, 
and  a  deep  abscess  scar  was  finally  formed.  If  the  sensitized 
animal  be  injected  intravenously  with  the  serum,  it  appears  rest- 
less, lies  on  its  belly,  and  respiration  frequently  increases;  it 
defecates  frequently,  finally  falls  upon  its  side,  and  commonly  dies, 
all  within  the  space  of  two  or  three  minutes. 

Serum  Sickness  in  Man. — Many  observers  have  noted  that  the 
injection  of  antitoxic  sera  into  man  sometimes  is  followed  by  a 
fever,  the  appearance  of  a  rash  or  urticaria,  pains  in  the  joints,  etc. 
In  some  individuals  the  reaction  is  shown  after  the  first  injection, 
in  most  cases  only  after  a  second  injection,  given  some  time  after 

176 


ANAPHYLAXIS   AND   HYPERSUSCEPTIBILITV  177 

the  first.     Evidently  there  may  be  an  inborn  or  a  developed  sen- 
sitiveness in  man  to  serum  injection. 

Theobald  Smith  Phenomenon. — Theobald  Smith  made  the 
observation  that  when  guinea-pigs  are  injected  with  horse  serum, 
and  a  second  injection  is  given  after  the  space  of  ten  days  or  more, 
the  pig  will  show  signs  of  hypersensitiveness,  and  if  a  sufficient 
quantity,  5  or  6  c.c.,  is  injected  intraperitoneally,  death  will  result 
in  a  few  minutes.  The  first  injection  serves  to  sensitize  against 
the  second.  This  phenomenon  in  the  guinea-pig  is  so  striking  that 
it  has  been  used  by  many  investigators  in  a  study  of  the  reaction. 
The  general  phenomenon  was  given  the  name  of  anaphylaxis. 
This  will  be  discussed,  and  its  utilization  in  diagnosis  and  in  ex- 
planation of  certain  hitherto  obscure  body  reactions  reviewed. 

Antibodies  in  Anaphylaxis. — Many  of  the  factors  determining 
anaphyla'xis  are  at  present  imperfectly  understood.  Enough  is 
known,  however,  to  enable  us*  to  account  for  many  hitherto 
obscure  body  reactions.  Various  species  of  animals  show  different 
types  of  anaphylactic  reaction,  but  the  differences  are,  according 
to  Anderson  and  Frost,  mainly  quantitative — the  nature  is  the 
same,  although  the  manifestations  are  different. 

Sensibilisinogen. — The  antigen  used  in  developing  anaphylaxis 
is  known  as  the  sensibilisinogen.  In  experimental  work  upon 
guinea-pigs  practically  all  proteins  have  been  found  to  sensitize, 
among  them,  blood-serum  of  many  animals,  egg-white,  milk, 
extracts  from  body  organs  and  cells,  plant  proteins,  bacterial 
proteins,  yeast  proteins,  and  even  some  of  the  peptones  formed 
by  the  peptic  digestion  of  proteins.  Experiments  have  shown  that 
the  sensibilisinogen  is  thermostabile;  in  many  cases  it  will  still 
sensitize  after  heating  to  a  temperature  above  boiling. 

Allergin. — The  injection  of  the  specific  protein  or  sensibili- 
sinogen has  been  shown  to  cause  the  production  in  the  blood  of  the 
guinea-pig  of  an  antibody  which  has  been  variously  termed 
allergin,  sensibilisin,  immune  body,  and  anaphylactin.  Either 
of  the  first  two  names  is  to  be  preferred  to  the  others.  That 
some  kind  of  an  antibody  is  formed  may  be  shown  by  injecting 
the  serum  of  a  sensitized  animal  into  a  normal  animal,  which  will 
then  show  the  anaphylactic  reaction  when  injected  with  the  proper 
sensibilisinogen.  Evidently  a  sensitizing  substance  (the  allergin) 
12 


178  VETERINARY  BACTERIOLOGY 

has  been  transferred  in  the  serum.  An  animal  sensitized  by  the 
injection  of  a  foreign  protein  or  sensibilisinogen  is  said  to  show 
active  anaphylaxis,  one  which  is  sensitized  by  the  serum  or  allergin 
of  another  sensitized  animal  is  said  to  show  passive  anaphylaxis. 
This  allergin  is  specific,  that  is,  an  animal  sensitized  to  egg-white 
will  not  show  anaphy lactic  reaction  when  injected  with  horse 
serum.  This  fact  might  be  utilized  in  testing  for  proteins  of 
different  kinds,  were  it  not  for  the  much  simpler  technic  of  the 
precipitation  reaction.  It  is  made  use  of,  as  will  be  noted  later,  in 
certain  disease  diagnoses.  How  this  allergin  is  formed  is  uncer- 
tain. Probably  the  primary  injection  of  the  protein  stimulates 
the  cells  of  the  body  to  increase  their  normal  power  of  assimilation. 
This  is  accomplished  by  certain  receptors,  which  break  up  the 
protein  (proteolysis) ,  or  so  change  it  that  it  can  be  rapidly  utilized 
by  the  cell.  These  receptors  increase  in  numbers,  and  are  thrown 
off  into  the  serum,  where  they  are  as  capable  of  transforming  the 
protein  when  injected  as  when  still  attached  to  the  protoplasm. 
The  allergin  retains  its  activity  after  heating  to  temperatures  of 
56°  to  58°;  it  is,  therefore,  regarded  by  Anderson  and  Frost  as 
thermostabile.  These  authors  believe  that  "  anaphy  lactic  shock 
is  due  to  disturbance  of  metabolic  activities  of  vital  cells  rather 
than  to  the  specific  (permanent)  intoxication  of  the  cells."  In 
other  words,  there  is  no  proof  that  the  substances  produced  from 
the  homologous  protein  (sensibilisinogen)  are  poisonous;  the  shock 
of  anaphylaxis  comes  from  the  disturbance  of  metabolism  of  the 
body-cells  due  to  the  sudden  flood  of  readily  assimilable  materials. 
Antianaphylaxis. — An  animal  which  has  just  recovered  from 
an  anaphylactic  shock  does  not  show  the  same  symptoms  when  a 
second  injection  is  made  soon  after.  Again,  if  a  small  injection 
be  made  into  a  sensitized  pig,  a  large  dose  a  few  hours  later  will 
fail  to  show  the  reaction.  The  possible  explanation  for  this 
phenomenon  has  been  offered,  to  the  effect  that  the  allergin  is  in 
each  case  all  used  up  by  the  first  injections,  and  the  second  injec- 
tion, therefore,  is  transformed  too  slowly  to  allow  any  reaction 
to  become  apparent.  Such  an  animal  is  said  to  be  in  a  state  of 
antianaphylaxis. 

Immunity. — Continued  injections  of  the  antigen  (sensibilisin- 
ogen) result  usually  in  the  development  of  an  immunity  in  the 


ANAPHYLAXIS   AND   HYPERSUSCEPTIBILITY  179 

experimental  animal.  Allergin  may  be  demonstrated  in  the  blood- 
serum  of  such  immunized  animals.  Immunity  must,  therefore, 
be  accounted  for  on  a  basis  other  than  antianaphylaxis.  It  may 
be  that  the  cells  no  longer  unite  with  the  products  of  the  action 
of  the  allergin  upon  the  antigen,  or  that  another  antibody  has  been 
developed  which  inhibits  the  action  of  the  allergin.  The  immune 
animal  is  only  relatively  susceptible,  and  may  be  shown  still  to 
be  somewhat  susceptible  by  appropriate  experimentation.  Ander- 
son and  Frost  state  "  anaphylaxis  is  a  step  toward  immunity, 
which  is  conceived  as  an  increased  capacity  for  safely  and  rapidly 
eliminating  the  specific  antigen  proteid." 

Relationship  of  Anaphylaxis  to  Certain  Body  Reactions.— 
The  anaphylactic  explanation  of  serum  sickness  in  man  has  already 
been  discussed.  Several  cases  are  on  record  where  injection  of 
diphtheria  antitoxin  has  resulted  in  death  within  a  few  minutes. 
Rosenau  and  Anderson  have  recorded  cases  in  which  the  prophylac- 
tic injection  of  antitoxic  serum  into  normal  individuals  resulted  in 
explosive  manifestations  of  anaphylaxis,  such  as  a  prickling  sensa- 
tion in  chest  and  neck,  labored  breathing,  paralysis,  convulsions, 
and  death  within  five  minutes  after  injection.  It  seems  evident 
that  the  individual  was  naturally  hypersensitive.  Fortunately, 
such  cases  are  extremely  rare.  The  prophylactic  and  curative 
value  of  diphtheria  antitoxin  far  outweigh  its  dangers. 

Rosenau  and  Anderson  have  also  recorded  some  evidence  that 
certain  of  the  toxemias  of  pregnancy,  particularly  puerperal 
eclampsia,  are  to  be  accounted  for  by  a  sensitization  of  the  body 
by  the  cells  of  the  placenta.  It  was  found  that  guinea-pigs  might 
be  sensitized  with  the  placenta  of  the  same  species  by  a  single 
injection,  and  a  second  injection  later  gave  a  typical  anaphylactic 
reaction. 

Bacterial  Anaphylaxis. — It  has  been  shown  by  several  inves- 
tigators that  proteins  from  bacteria  may  be  used  in  sensitizing 
animals  against  a  second  injection;  in  other  words,  they  resemble 
proteins  from  other  sources  in  this  respect.  Extracts  from  Bacillus 
coli,  B.  anthracis,  B.  tuberculosis,  B.  typhosus,  and  others  have  been 
shown  to  sensitize.  This  fact  has  been  deemed  of  considerable 
practical  importance  in  disease  diagnosis.  There  is  reason  to 
suppose  that  infection  with  certain  bacteria  will  sensitize  the  body 


180  VETERINARY   BACTERIOLOGY 

to  the  injection  of  the  bacterial  proteins  or  extracts  or  to  the  dead 
bodies  of  the  organism.  It  has  been  found,  for  example,  if  the  dead 
organisms,  or  extracts  from  them,  be  rubbed  into  the  skin  of  an 
infected  individual,  an  inflammation,  marked  by  some  edema, 
redness,  and  frequently  the  formation  of  papules,  will  occur. 
This  is  true  in  tuberculosis  and  some  other  diseases.  That  this  is 
one  of  the  manifestations  of  anaphylaxis  seems  probable.  The 
injection  of  tuberculin,  consisting  of  dead  tubercle  bacilli  and  their 
products  of  growth,  into  an  animal  having  tuberculosis  will  cause 
a  characteristic  rise  in  temperature,  and  is,  therefore,  of  great 
diagnostic  value.  This  reaction  resembles  the  anaphylactic 
reaction  in  many  ways.  For  example,  a  second  injection  following 
soon  after  the  first  will  give  no  reaction;  probably  the  body  is 
in  a  state  of  antianaphylaxis.  Furthermore,  advanced  cases 
of  the  disease  do  not  respond — perhaps  they  are  either  constantly 
antianaphylactic  or  show  anaphylactic  immunity  due  to  the 
constant  presence  of  large  numbers  of  organisms  in  the  body. 
On  the  other  hand,  there  are  some  unexplained  differences  between 
this  reaction  and  that  typical  of  anaphylaxis  produced  by  other 
proteins.  Many  points  remain  still  to  be  explained.  That  it  is, 
however,  of  a  similar  nature  seems  to  be  altogether  probable.  A 
similar  reaction  may  be  secured  with  the  emulsion  of  dead  Bacillus 
mallei  (mallein)  in  glanders. 


CHAPTER  XIX 

AGGRESSINS 

THE  term  virulence,  as  applied  to  microorganisms,  is  not 
readily  defined.  The  reaction  of  an  organism  in  the  body  cannot 
be  predicted  by  its  behavior  in  culture-media.  The  most  virulent, 
diphtheria  bacillus,  for  example,  is  not  necessarily  the  one  that 
produces  the  largest  amounts  of  toxin  in  culture-media.  Con- 
cerning this  mechanism  of  virulence  there  have  been  much  dis- 
cussion and  speculation. 

Bail  has  developed  what  he  has  termed  an  aggressin  hypothesis 
to  account  for  these  variations  in  virulence.  He  has  defined  an 
aggressin  as  a  substance  which  is  secreted  by  pathogenic  organisms, 
when  growing  in  the  animal  body,  which  will  neutralize  the  efforts 
of  the  tissues  to  destroy  the  organism.  The  work  on  this  subject 
has  been  done  principally  by  Bail  and  his  students,  although  many 
substantiating  facts  have  been  developed  by  other  investigators. 
The  theory  is  of  practical  importance,  inasmuch  as  it  is  claimed 
that  the  injection  of  bacteria-free  aggressins  into  an  animal  results 
in  the  ultimate  development  of  antiaggressin  and  of  an  active 
immunity. 

Bail  divides  bacteria  into  three  groups — true  parasites,  half 
parasites,  and  saprophytes.  The  true  parasites  include  such 
forms  as  the  anthrax  bacillus,  in  which  the  injection  of  a  very 
small  number  results  fatally  through  a  rapid  multiplication  of  the 
organisms.  The  half  parasites  are  those  having  a  lower  degree 
of  virulence — such  that  relatively  large  injections  must  be  made  to 
produce  infection.  The  third  group  includes  those  totally  devoid 
of  pathogenic  properties.  The  difference  between  these  forms  is  a 
difference  in  the  ability  to  produce  aggressin. 

Aggressin  production  is  demonstrated  by  Bail  as  follows: 
A  guinea-pig  is  injected  intraperitoneally  with  many  times  a  fatal 
dose  of  culture  of  Bacillus  typhosus  or  Spirillum  cholerce.  The 

181 


182  VETERINARY   BACTERIOLOGY 

peritoneal  exudate  is  removed  after  the  death  of  the  animal  and 
centrifuged  to  remove  the  cellular  elements  as  well  as  most  of  the 
bacteria.  The  organisms  remaining  in  the  supernatant  liquid  are 
destroyed  by  a  chemical  disinfectant.  If  a  small  quantity  of  this  is 
injected  into  a  guinea-pig,  together  with  a  quantity  of  the  specific 
organisms  that  would  usually  be  non-lethal,  the  animal  will  die' 
with  an  acute  infection.  The  injected  exudate  apparently  makes 
the  organisms  more  virulent.  The  aggressins  are  contained  in 
this  exudate,  and  convert  a  mild  infection  into  one  that  is  fatal. 
The  repeated  injection  of  sterile  aggressin  causes  the  development 
in  the  animal  body  of  an  active  immunity.  According  to  Bail, 
antiaggressins  can  be  demonstrated  in  the  blood-serum  of  such 
animals.  One  of  the  strongest  links  of  corroborative  evidence 
is  the  fact  that  the  bacteriolytic  power  of  a  serum  in  vitro  is  not  an 
index  of  the  ability  of  the  animal  from  which  it  was  taken  to  resist 
infection.  The  modus  operandi  of  the  aggressin  is  to  be  sought  in 
the  inhibition  of  phagocytosis.  Bail  found  that  when  the  typhoid 
bacillus  is  injected  intraperitoneally,  there  is  a  marked  increase  in 
the  number  of  phagocytes  in  the  exudate  within  a  few  hours,  but 
when  they  are  injected  with  aggressin,  there  is  no  such  increase — 
the  phagocytes  are  evidently  not  attracted. 

This  theory  has  not  been  accepted  in  its  entirety  by  many 
bacteriologists.  The  facts  given  by  Bail  are  largely  accepted, 
but  the  theory  is  not  well  established.  Some  authors  claim  to  have 
shown  that  the  so-called  aggressin  is  simply  endotoxin  released  in 
the  body  by  the  lysis  of  the  bacterial  cells.  It  is  also  claimed  that 
there  is  no  demonstrable  difference  between  the  endotoxins  pro- 
duced in  culture-media  and  the  aggressins  formed  in  the  body. 
Peritoneal  exudate  containing  aggressin,  according  to  Bail,  has  been 
filtered  through  porcelain,  and  found  to  be  toxic  for  guinea-pigs 
after  filtration.  Bail  has  attempted  to  develop  practicable  methods 
of  immunization  in  several  diseases  by  an  application  of  the  theory. 
None  of  his  methods  have  come  into  general  use. 


SECTION    IV 

PATHOGENIC  MICROORGANISMS,  EXCLUSIVE  OF  THE 

PROTOZOA 


CHAPTER  XX 

MICROORGANISMS  AS  A  CAUSE  OF  DISEASE 

Infectious  Diseases. — An  infectious  disease  is  one  caused  by  a 
microorganism.  In  general,  the  presence  of  microorganisms  in  the 
body  does  not  constitute  infection  unless  proliferation  and  mul- 
tiplication occurs  and  there  are  pathological  changes  in  tissues. 
An  individual  harboring  such  an  organism  is  called  the  host, 
and  is  said  to  be  infected.  The  latter  expression  is  commonly 
used  also  to  describe  any  object,  such  as  a  surgical  instrument, 
which  may  introduce  pathogenic  organisms  into  the  body;  a  better 
term  for  such,  however,  is  infective.  An  infective  object,  such  as 
the  clothing  of  a  diphtheritic  patient  or  the  manger  of  a  glandered 
horse,  is  sometimes  called  a  fomite.  An  organism  which  will 
produce  disease  is  sometimes  spoken  of  as  a  virus;  it  is  customary 
to  speak  of  the  cause  of  a  disease  where  the  infecting  agent  is 
not  known  by  this  name.  By  no  means  all  diseases  are  infectious. 
Among  the  non-infectious  diseases  may  be  named  azoturia,  dia- 
betes, Bright's  disease,  etc. 

Contagious  Diseases. — Any  disease  in  which  the  causal  organ- 
ism may  be  readily  transferred  from  one  individual  to  another  by 
direct  or  indirect  contact  is  said  to  be  contagious.  The  terms  in- 
fectious and  contagious  have  been  used  very  loosely,  particularly 
in  veterinary  writings,  sometimes  even  interchangeably.  The 
best  usage,  however,  is  strictly  to  limit  the  terms  as  here  defined. 
Infectious  has  to  do  with  the  cause  of  a  disease,  and  contagious 
with  its  ease  and  method  of  transmission.  All  infectious  diseases, 

183 


184  VETERINARY   BACTERIOLOGY 

therefore,  which  are  readily  transmitted  by  contact,  direct  or 
indirect,  are  said  to  be  contagious.  All  contagious  diseases  are 
necessarily  infectious,  but  the  reverse  is  not  true;  for  example, 
malaria  in  man  and  Texas  fever  in  cattle  cannot  be  regarded  as 
contagious,  as  they  are  transmitted  only  through  the  bite  of  cer- 
tain insects. 

Bacteria  Normally  Present  in  the  Body  or  on  its  Surface. — 
Many  microorganisms  are  found  in  the  healthy  body.  In  some 
cases  they  are  harmless  commensals;  in  others,  they  merely  await 
a  favorable  opportunity  to  invade  the  tissues.  The  care  used  in 
surgical  operations  is  made  necessary  by  the  presence  of  these 
organisms. 

Bacteria  of  the  Skin. — Some  bacteria  are  found  quite  constantly 
upon  the  skin  and  hair,  others  may  be  isolated  only  occasionally. 
Staphylococci  and  streptococci  are  generally  present,  and  many 
of  them  are  capable  of  causing  pus  infection  when  introduced 
under  the  skin.  Certain  organisms,  such  as  the  Bacillus  smegmatis, 
are  found  commonly  in  the  axillae,  where  the  skin  is  moist  and 
frequently  greasy. 

Bacteria  of  the  Mouth. — Microorganisms  to  the  number  of  sixty- 
two  species  were  found  by  Miller  in  the  human  mouth.  Of  these 
he  found  ten  quite  constantly  present.  The  flora  of  the  mouth  in 
domestic  animals  has  not  been  accurately  determined,  but  large 
numbers  of  bacteria  are  always  present.  Some  of  these  are 
pyogenic  cocci. 

Bacteria  of  the  Stomach. — The  acidity  of  the  gastric  juice  in 
man  destroys  many  of  the  microorganisms  which  enter  the  stomach. 
In  cases  where  the  normal  acidity  is  absent  many  bacteria  will 
multiply.  The  flora  of  the  stomach  in  animals  is  not  well  under- 
stood. Bloat  is  due  to  the  development  of  gas-producing  bacteria 
in  the  ingested  carbohydrate-rich  food.  The  organisms  respon- 
sible are  probably  those  normal  to  the  intestinal  tract. 

Bacteria  of  the  Small  Intestine  and  Colon. — The  flora  of  the 
intestines  in  man  has  been  studied  at  length  by  bacteriologists, 
but  that  of  animals  is  not  so  well  understood.  The  organisms 
most  common  are  those  belonging  to  the  so-called  intestinal 
group,  such  as  Bacillus  coli  and  Bacillus  lactis  aerogenes.  Strep- 
tococci are  generally  present  in  feces.  Certain  putrefactive 


MICROORGANISMS   AS   A    CAUSE   OF  DISEASE  185 

anaerobes,  such  as  Bacillus  putrificus,  are  sometimes  found  in  large 
numbers.  Amebse  and  certain  other  protozoan  types  are  fre- 
quently found.  The  intestinal  juices,  particularly  the  bile,  in- 
hibit the  growth  of  some  species,  but  the  normal  inhabitants  of 
the  digestive  tract  will  grow  and  multiply  in  pure  bile.  The 
number  of  bacteria  increases  from  the  stomach  on,  the  greatest 
numbers  being  found  in  the  colon.  Here  the  living  bacterial 
cells  are  frequently  present  by  hundreds  of  millions  to  the  gram. 
It  has  been  estimated  that  human  feces  sometimes  contain  as 
much  as  38  per  cent,  of  their  bulk  in  bacteria.  The  bulk  in 
herbivorous  animals  is  certainly  much  lower.  Some  of  these 
bacteria  are  harmless  commensals,  but  a  few  are  believed  sometimes 
to  assume  pathogenic  roles.  It  has  been  claimed  that  in  the  case 
of  herbivora  certain  of  these  bacterial  cells  secrete  cellulase  and 
assist  materially  in  the  digestion  of  cellulose.  This  has  not  been 
satisfactorily  demonstrated,  but  is  not  improbable.  Whether  or 
not  bacteria  in  the  intestines  are  essential  to  the  maintenance  of 
health  is  a  mooted  question.  Experimental  evidence  is  conflict- 
ing. Practically,  infection  of  the  intestinal  tract  occurs  very  early 
in  life.  Metchnikoff  and  his  school  have  claimed  that  in  man  some 
of  the  symptoms  of  old  age,  such  as  arteriosclerosis,  are  due  to  the 
absorption  of  poisonous  products  of  bacterial  putrefaction,  and 
has  sought  to  establish  an  intestinal  flora  consisting  of  non-putre- 
factive types. 

Bacteria  of  the  Organs  of  Respiration. — Air  entering  the  nose 
is  rapidly  freed  from  dust  and  bacteria  by  contact  with  the  moist 
surfaces  of  the  lining  membranes,  consequently  the  nose  may  have 
many  types  of  organisms  present.  It  is  a  most  efficient  filtering 
device,  so  that  very  few  bacteria  gain  entrance  to  the  trachea  and 
the  lungs,  and  these  are  generally  quickly  eliminated. 

Bacteria  of  the  Genito-urinary  Organs. — The  secretions  of  the 
vagina  seem  effectually  to  prevent  bacterial  growth.  The  uterus 
is  normally  bacteria  free,  as  is  also  the  bladder.  The  urethra 
contains  few  organisms. 

Avenues  of  Infection. — The  avenue  through  which  an  organism 
gains  entrance  to  the  body  is  called  its  portal  of  entry  or  its  infec- 
tion atrium.  An  infection  arising  from  contact  with  infective 
external  objects  is  termed  exogenous;  one  caused  by  organisms 


186  VETERINARY  BACTERIOLOGY 

constantly,  or  normally  present  in  the  body  or  on  it  is  termed  endog- 
enous. Traumatic  infection  frequently  occurs  through  a  break 
in  the  continuity  of  the  skin  or  mucous  surfaces.  Wounds  caused 
by  weapons,  instruments,  and  similar  objects,  the  bites  of  animals 
and  sucking  insects  such  as  the  mosquito,  flea,  tick,  or  bed-bug, 
may  introduce  a  pathogenic  organism.  Ordinarily,  the  skin  is  an 
efficient  barrier  against  infection.  Microorganisms  may  occa- 
sionally enter  through  the  glands  or  hair  follicles.  Some  bacteria 
apparently  may  injure  the  unbroken  mucous  surface,  as,  for  ex- 
ample, the  diphtheria  bacillus.  Certain  disease  organisms  enter 
through  the  digestive  tract.  Bacteria  have  been  shown  to  pass 
unharmed  through  the  intestinal  walls  and  to  enter  the  lymph- 
vessels  and  the  thoracic  duct.  To  what  extent  this  is  the  common 
source  of  infection  in  certain  diseases,  such  as  tuberculosis,  is  at 
present  a  matter  of  dispute. 

The  lungs  constitute  the  infection  atrium  in  pneumonia, 
probably  in  many  cases  of  tuberculosis  and  aspergillosis,  and  pos- 
sibly in  certain  diseases  whose  cause  has  not  been  determined, 
such  as  small-pox. 

The  genital  organs  are  the  common  infection  atria  in  the 
so-called  venereal  diseases,  such  as  syphilis,  chancroid,  and  gonor- 
rhea in  man,  dourine  in  the  horse,  and  contagious  abortion  in 
cattle.  Disease  organisms  rarely  pass  from  the  blood  of  the 
mother  through  the  placenta  to  the  blood  of  the  fetus.  There 
are  comparatively  few  diseases,  therefore,  that  are  inheritable. 
Syphilis  and  small-pox  are  exceptions. 

Types  of  Disease  Produced  by  Microorganisms. — The  char- 
acteristics of  the  disease  produced  by  various  microorganisms,  and 
the  pathological  changes  they  bring  about,  vary  as  much  as  do  the 
morphological  and  cultural  characters  of  the  organisms  themselves. 
It  is  possible,  however,  to  group  related  types  together  and  find 
a  general  basis  for  classification. 

Specific  and  Non-specific  Infections. — A  specific  infectious 
disease  is  one  caused  in  every  case  by  a  single  species  of  organism, 
and  characterized  by  definite  clinical  characters  or  symptom- 
complex  and  pathological  lesions.  A  non-specific  infection  is  one 
which  may  be  caused  by  one  of  several  organisms,  and  does  not 
possess  the  definite  characters  which  enable  the  determination  of 


MICROORGANISMS   AS   A   CAUSE    OF   DISEASE  187 

the  specific  cause  by  clinical  examination  or  from  the  general 
character  of  the  lesions  produced.  Such  infections  as  those 
produced  by  the  common  pyogenic  bacteria  are  non-specific. 

Primary,  Secondary,  and  Mixed  Infections. — Any  disease  caused 
by  a  single  organism  may  be  termed  a  primary  infection.  One 
organism  may  break  down  the  body  defenses  and  make  infection 
with  another  and  second  species  easy.  The  occurrence  of  pneu- 
monia as  a  sequel  to  measles  in  man,  for  example,  would  be  termed 
a  secondary  infection.  Two  or  more  organisms  associated  in 
bringing  about  changes  are  said  to  cause  a  mixed  infection,  as  is 
frequently  the  case  in  wound  suppuration. 

Classification  of  Disease  Types. — Certain  disease  organisms  do 
not  frequently  enter  the  general  circulation,  but  proliferate  in  a 
more  or  less  circumscribed  area.  In  some  cases  such  an  organism 
produces  toxins,  which  are  absorbed  into  the  blood-stream  and 
cause  injury  to  remote  tissues,  as  in  diphtheria  and  tetanus. 
Such  a  disease  is  called  a  toxemia.  When,  on  the  other  hand,  no 
appreciable  amount  of  toxin  is  produced,  it  is  known  as  a  phlo- 
gistic disease  or  infection,  such  as  wound  suppuration  and  gonor- 
rhea. A  general  invasion  of  the  blood-stream  is  called  a  bacter- 
emia.  The  term  septicemia  has  come  to  be  used  synonymously 
with  bacteremia,  although  more  correctly  applied  to  those  types 
produced  by  pyogenic  organisms.  Sapremia  is  produced  by  the 
absorption  of  poisonous  putrefactive  products  or  the  destruction 
of  necrotic  tissues  by  saprophytic  bacteria.  Pyemia  is  a  metastatic 
pyogenic  infection.  Diseases  characterized  by  skin  eruptions  of 
certain  types  are  called  exanthemata  (sing,  exanthema}.  For  the 
most  part,  the  causes  of  these,  such  as  small-pox,  chicken-pox, 
sheep-pox,  scarlet  fever,  etc.,  have  not  been  certainly  determined. 

How  Bacteria  Produce  Disease. — A  few  bacteria  produce  the 
specific  toxins  which  have  already  been  discussed.  Intracellular 
poisons,  called  endotoxins,  are  produced  by  other  forms,  and  are 
freed  only  by  the  dissolution  of  the  cell.  Possibly,  disease  may 
sometimes  be  produced  by  mechanical  means.  The  mechanism 
in  other  cases  is  imperfectly  understood. 

Groups  of  Pathogenic  Microorganisms,  Exclusive  of  Protozoa. 
— The  disease-producing  organisms  can  best  be  studied  after  a 
classification  of  the  related  forms  into  groups.  The  characters 


188  VETERINARY    BACTERIOLOGY 

belonging  in  common  to  all  the  organisms  of  a  group  are  more 
easily  remembered  than  when  learned  separately  for  each  species. 
A  classification  may  be  based  upon  the  character  of  the  disease 
produced;  that  is,  be  pathologic.  Such  a  classification  is  open 
to  the  objection  that  very  different  types  of  infection  may  be  pro- 
duced under  different  conditions  by  the  same  or  closely  related 
organisms.  Certain  pus  cocci,  for  example,  may  produce  erysip- 
elas, wound  suppuration,  septicemia,  pyemia,  or  local  infection, 
and  inflammation  of  almost  any  organ  of  the  body.  On  the  other 
hand,  infections  having  similar  clinical  characters  may  be  pro- 
duced by  very  different  organisms.  A  bacteriologic  classification 
is  based  upon  resemblances  in  morphologic,  physiologic,  and  cul- 
tural characters. 

The  following  classification,  in  the  main  bacteriologic,  will  be 
used  in  the  differentiation  of  the  various  groups  of  known  micro- 
organisms, exclusive  of  the  protozoa,  that  are  of  pathogenic 
veterinary  interest. 


Principal  Groups  of  Pathogenic  Microorganisms 

I.  True  bacteria. 

A.  Cells  spherical,  cocci. 

1.  Non-specific    pyogenic    organisms.     Non-specific   pyogenic  coccus 
group  (1). 

2.  Causing  specific  infections.     Specific  coccus  group  (2). 

B.  Cells  rod-shaped.     Bacilli. 

1.  Non-specific    pyogenic     forms.     Non-specific    pyogenic    bacillus 
group  (3). 

2.  Usually  associated  with  specific  infections, 
a.  Aerobic  or  facultative  anaerobic. 

(1)  Non-spore  producing, 
(a)  Not  acid  fast. 

+  Gram  positive. 

1.  Diphtheria  group  (4). 

2.  Bacillus  pseudotuberculosis  group  (5). 

3.  Swine  erysipelas  group  (6). 
+  +  Gram  negative. 

1.  Glanders  group  (7). 

2.  Intestinal  group  (8). 

3.  Hemorrhagic  septicemia  group  (9). 

4.  Fowl  diphtheria  group  (10). 
(6)  Acid  fast.     Acid  fast  group  (11). 

(2)  Spore  producing.     Anthrax  group  (12). 


MICROORGANISMS   AS   A   CAUSE   OF   DISEASE  189 

b.  Anaerobic  or  microaerophilic. 

(1)  Non-spore  producing. 

Short  bacillus  type.     Abortion  bacillus  group  (13). 
Long  slender  beaded  rods.     Necrosis  bacillus  group  (14). 

(2)  Spore  producing.     Anaerobic  spore-producing  group  (15). 

C.  Cells  spiral.     Spirilla. 

1.  Spirillum  or  Asiatic  cholera  group  (16). 

2.  Spirochaete  group  (17). 1 

D.  Cells  elongate,  forming  threads,  branched.     Actinomyces  group  (18). 

II.  Yeasts  or  yeast-like  forms.     Blastomyces  group  (19). 

III.  Molds  or  mold-like  forms.   >Hyphomycete  group  (20). 

1  The  spirochaetes,  on  account  of  their  protozoan  resemblances,  are  grouped 
with  the  protozoa  and  not  the  bacteria. 


CHAPTER  XXI 

NON-SPECIFIC  PYOGENIC  COCCI 

THE  non-specific  pyogenic  cocci  are  characteristic  of  wound 
infections,  suppuration,  non-specific  inflammations,  and  their 
sequelae.  A  considerable  number  of  microorganisms  have  been 
described  belonging  to  this  group.  They  are  found  quite  com- 
monly upon  the  surface  of  the  skin.  In  pathogenicity,  they  vary 
from  non-virulent  strains  to  those  which  will  kill  experimental 
animals  promptly  when  introduced  in  minute  doses.  Even  the 
same  organism  may  be  made  to  vary  its  virulence  by  proper 
cultural  methods. 

An  organism  which  is  capable  of  causing  suppuration  or  pus 
production  is  said  to  be  pyogenic.  A  long  list  of  organisms  are 
known  which  can  bring  about  this  tissue  reaction,  but  the  coccal 
forms  to  be  studied  are  by  far  the  most  common  in  suppurative 
processes  in  man  and  animals.  Whenever  these  organisms  invade 
the  tissues,  or  are  introduced  through  a  wound,  they  begin  to 
multiply  and  to  destroy,  and  seemingly,  to  some  extent,  to  dis- 
integrate the  tissues  with  which  they  are  in  contact.  The  body 
reacts,  in  general,  by  an  inflammation  of  the  surrounding  tissues, 
the  blood-vessels  become  dilated,  there  is  more  or  less  extravasa- 
tion of  blood-serum.  Most  important  of  all,  the  phagocytic 
white  blood-cells,  particularly  the  polymorphonuclear  leukocytes, 
pass  out  of  the  capillaries  in  great  numbers,  so  that  the  tissues 
become  packed  full  and  the  lesion  is  surrounded  by  a  phagocytic 
wall,  which  usually  effectually  prevents  the  bacteria  from  spread- 
ing. The  leukocytes  also  invade  the  diseased  tissue  and  eventually 
destroy  the  bacteria.  This  can,  of  course,  be  accomplished  only 
in  the  presence  of  opsonins.  Nor  is  it  brought  about  without 
a  struggle,  for  many  of  the  leukocytes  themselves  are  destroyed  by 
the  bacteria,  possibly  through  the  leukocytotoxic  substances 

190 


NON-SPECIFIC    PYOGENIC   COCCI  191 

produced  by  them.  The  mixture  of  blood-serum,  white  blood- 
cells,  bacteria,  and  disintegrated  tissue  is  called  pus. 

Until  recent  times  it  was  supposed  that  no  wound  could 
heal  normally  and  naturally  without  pus  being  produced;  it  was 
regarded  as  a  more  or  less  essential  step  in  the  healing  process. 
When  it  was  discovered  that  pus  production  was  the  result  of 
infection,  and  that  healing  by  first  intention  (without  pus  formation) 
was  desirable,  every  effort  was  made  to  disinfect  the  wounds 
made  in  surgical  operations.  This  antiseptic  surgery  was  un- 
questionably an  advance  over  that  previously  practised,  but  the 
use  of  the  strong  disinfecting  solutions  irritated  the  tissues.  At 
present  the  surgeon  uses  every  precaution  to  prevent  the  entrance 
of  extraneous  bacteria  into  the  wound,  and  dependence  is  placed 
upon  the  natural  resistance  of  the  body  and  its  immunizing 
agencies  to  prevent  the  development  of  those  organisms  which 
cannot  be  removed  from  the  skin.  This  has  been  termed  aseptic 
surgery. 

While  these  organisms  are  usually  associated  with  local  inflam- 
mation and  suppuration,  they  may  gain  entrance  to  the  blood- 
stream and  produce  septicemia,  pyemia,  and  metastatic  infections 
in  many  organs  and  tissues. 

Organisms  Belonging  to  this  Group. — The  organisms  belonging 
to  this  group  may  be  subdivided  into  the  Micrococci  (Staphylo- 
cocci)  and  Streptococci.  The  following  species  will  be  discussed: 
Micrococcus  aureus,  M.  albus,  M.  citreus,  M.  bovis,  M.  mastitidis, 
M.  ovis,  M.  epidermidis  albus,  M.  cereus  albus,  M.  cereus  flavus, 
Streptococcus  pyogenes,  and  the  closely  related  Streptococcus 
lacticus.  The  student  must  expect  to  find  in  bacteriologic  and 
pathologic  literature  the  greatest  diversity  of  treatment  of  these 
forms.  It  is  important  that  the  various  synonyms  of  the  names  of 
the  more  important  organisms  should  be  learned,  that  they  may  be 
recognized  hereafter.  For  example,  the  term  Staphylococcus  will 
be  found  in  literature  quite  as  commonly  as  the  form  Micrococcus, 
which  is  here  used.  It  will  be  well  before  beginning  the  study  of 
the  specific  bacteria  to  read  again  the  chapter  on  Classification  of 
Microorganisms. 

Common  Characters. — The  organisms  belonging  to  this  group 
resemble  each  other  in  being  cocci,  without  spores,  non-motile, 


192 


VETERINARY   BACTERIOLOGY 


and  gram-positive.  They  are  all  aerobic  and  facultative  anaerobic, 
and  all  grow,  though  in  some  cases  poorly,  upon  most  of  the  com- 
mon laboratory  media. 

Micrococcus  aureus 

Synonyms.  —  Micrococcus  pyogenes  aureus;  Staphylococcus  pyog- 
enes  aureus;  Staphylococcus  aureus. 

Micrococci  were  definitely  described  as  present  in  pus  by  Ogston 
(1881).  Three  years  later  Rosenbach  (1884)  cultivated  them  upon 
artificial  media  and  differentiated  several  species,  among  them  the 
one  under  consideration.  Other  investigators  have  frequently 


Fig.  82.  —  Stained  mount  of  the 
Micrococcus  aureus  from  agar  (Gun- 
ther). 


Fig.   83.  —  Colony    of    Micrococcus 
aureus  on  agar  (Heim). 


isolated  this  organism  from  pus  in  man  and  practically  all  domestic 
animals. 

Distribution  in  Nature.  —  Micrococcus  aureus  occurs  quite  con- 
stantly upon  the  skin  and  hair  of  man  and  animals,  in  the  nose 
and  mouth  of  man,  occasionally  in  human  feces,  and  frequently 
in  milk.  It  is  often  carried  about  in  atmospheric  dust,  and  is 
not  uncommon  in  water,  especially  when  contaminated  with 
sewage. 

Morphology  and  Staining  Characters.  —  The  organism  is 
spherical,  sometimes  slightly  flattened  where  two  are  appressed,  and 
in  pus  and  in  the  blood  is  usually  in  masses  like  grape-clusters 
(whence  the  name,  Staphylococcus)  ;  in  culture-media  the  cells  are 


NON-SPECIFIC   PYOGENIC  COCCI  193 

grouped  irregularly.  The  diameter  of  the  cells  is  about  0.7  to 
0.9  u.,  rarely  larger.  It  stains  well  with  ordinary  anilin  dyes  and 
is  gram-positive. 

Isolation  and  Culture. — Micrococcus  aureus  may  be  frequently 
secured  in  pure  culture  by  making  cultures  directly  from  a  fistula 
or  other  suppurating  focus,  first  cleansing  the  outer  portion  and 
securing  material  on  a  sterile  platinum  needle.  In  general  it  is 
best,  however,  to  plate  out  the  drop  of  pus  obtained  in  this  way. 
This  is  not  only  important  in  preventing  contamination,  but 
hi  order  to  diagnose,  mixed  infections,  especially  in  isolations  made 
for  the  purpose  of  preparing  autogenic  vaccines.  The  organism 
grows  well  in  all  the  common  laboratory  media.  The  colonies 
upon  gelatin  appear  as  disks  with  smooth,  definite  edges,  and  with 
granular,  dark  interior.  Within  a  few  days  the  colony  sinks  in  a 
cup  of  liquid.  The  liquid  is  cloudy,  with  a  golden-yellow  sediment. 
The  growth  on  agar  is  abundant,  shining,  and  well  circumscribed. 
Upon  the  potato  the  growth  is  luxuriant,  and  the  orange  pigment 
is  produced  here  in  greatest  abundance.  Bouillon  is  clouded. 
Milk  is  curdled  with  a  slight  acid  reaction,  and  the  curd  is  eventu- 
ally digested. 

Physiology. — A  study  of  the  physiologic  characters  of  this 
organism  makes  it  apparent  that  there  are  many  races  of  which 
account  must  be  taken.  It  has  not  been  satisfactorily  demon- 
strated, however,  that  there  is  any  relationship  between  these 
variations  and  pathogenicity. 

Pigment  Production. — An  orange-yellow  pigment  is  produced. 

Fermentation. — A  slight  power  to  produce  acid  in  milk  has  been 
noted.  No  gas  is  produced.  Nitrates  are  reduced  to  nitrites. 
A  proteolytic  enzyme  which  digests  casein  is  produced  in  milk. 
Gelatinase  is  found  in  gelatin.  Rennin  and  maltase  have  been 
demonstrated. 

Relation  to  Oxygen. — The  organism  is  aerobic  and  facultative 
anaerobic. 

Vitality. — There  is  a  marked  resistance  to  desiccation,  although 
it  is  not  probable  that  the  organism  can  remain  alive  for  very  long 
periods  in  this  condition.  Cultures  upon  media  will  remain  alive 
for  months.  The  optimum  growth  temperature  is  blood-heat, 
although  growth  is  good  at  room-temperatures  and  below.  The 

13 


194  VETERINARY    BACTERIOLOGY 

various  isolated  strains  have  shown  great  variation  in  heat  resis- 
tance. Usually  a  temperature  of  60°  for  half  an  hour  suffices  to 
destroy  all  the  cells,  but  some  require  80°  for  the  same  length  of 
time.  The  cells  are  easily  destroyed  by  common  disinfectants. 

Pathogenesis. — Mechanism  of  Disease  Production. — Staphylo- 
cocci  have  been  shown  to  produce  a  leukocytotoxin  called  leuko- 
cidin.  A  stained  mount  of  pus  will  frequently  show  that  many  of 
the  leukocytes  have  been  destroyed,  and  that  the  cells  are  begin- 
ning to  disintegrate.  Staphylolysin,  a  hemolytic  toxin,  is  also 
produced,  particularly  by  the  virulent  strains.  It  seems  to  have 
been  demonstrated,  however,  that  these  two  toxins  do  not  explain 
the  pathogenic  nature  of  the  organism  satisfactorily.  Possibly 
anaphylaxis  will  explain  some  of  the  reactions  secured,  but  there 
seems  to  be  some  poisonous  property,  possibly  an  endotoxin, 
which  is  not  at  present  understood.  We  have  no  satisfactory 
explanation  for  its  mechanism  of  disease  production. 

Experimental  Evidence  of  Pathogenesis. — The  causal  relation- 
ship of  this  organism  to  pus-production  and  wound  infection  has 
been  amply  demonstrated.  One-tenth  c.c.  of  a  twenty-four-hour 
culture  of  a  moderately  virulent  strain  will  kill  a  rabbit,  when 
injected  intravenously,  in  four  to  eight  days,  and,  upon  post- 
mortem examination,  abscesses  containing  the  same  organism  will 
be  found  in  many  of  the  internal  organs. 

Types  of  Natural  Infection. — As  has  been  stated  previously, 
Micrococcus  aureus  is  most  frequently  found  associated  with 
wound  infections,  and  is  also  the  common  cause  of  abscesses, 
carbuncles,  boils,  acne,  and  furuncles  in  man  and  animals,  of 
poll-evil  and  fistula  in  the  horse,  and  similar  lesions  in  other  animals. 
The  organism  may  gain  entrance  to  the  circulation  and  produce 
septicemia  or  pyemia  in  man,  rarely  in  animals.  It  has  also  been 
found  in  man  as  the  cause  of  certain  metastatic  infections,  par- 
ticularly of  the  bone-marrow  (osteomyelitis),  and  ulcerative 
endocarditis.  These  have  also  been  produced  experimentally  in 
the  laboratory  animals.  Inflammation  of  the  udder  in  cows 
(mastitis)  is  occasionallly  caused  by  this  organism. 

Immunity. — The  production  of  two  specific  toxins,  the  hemo- 
lytic staphylolysin  and  the  leukocidin,  has  already  been  noted, 
as  also  the  probable  importance  of  an  endotoxin.  Antitoxins 


X(  IN-SPECIFIC   PYOGEXIC   COCCI 


195 


for  the  two  first  mentioned  have  been  produced,  but  do  not  seem 
to  confer  immunity.  Agglutinins  have  been  demonstrated  in 
normal  as  well  as  infected  animals;  they  seem,  however,  to  be  of 
no  diagnostic  value.  The  precipitation  reaction  has  likewise 
been  obtained  with  the  bacterial  filtrate.  Bail  has  claimed  that 
the  production  of  aggressins  accounts  for  pathogenicity,  but  his 
results  are  inconclusive.  Bacteriolysins  for  Micrococcus  aureus 
are  not  produced  in  appreciable  quantities,  and  are  probably  not 
of  any  immunizing  value.  Opsonins  may  be  demonstrated  in 
both  normal  and  immune  blood,  and  seem  to  be  the  most  important 
serum  component  in  determining  immunity. 

Immunization. — Bacterins  and  vaccines,  both  univalent  and 
polyvalent,  have  been  prepared,  and  have  been  extensively  tested 
in  cases  of  chronic  sup- 
puration. The  results 
of  repeated  injections 
have  been  encouraging 
in  many  cases.  A  check 
has  been  kept  on  the 
development  of  immu-  pi 
nity  in  man  by  repeated 
determinations  of  the 
opsonic  index.  Care  is 
used  to  eliminate  the 
negative  phase  as  far  as 
possible  in  making  the 
injections.  Autogenic 
vaccines  have  proved  Fig  8^_3fiamamu  aureus  in  pus  (Frankel 
even  more  successful  in  and  Pfeiffer). 

both  man  and  animals. 

The  methods  of  preparation  of  such  a  vaccine  have  already  been 
discussed.  It  seems  altogether  likely  that  the  use  of  bacterins 
and  of  autogenic  vaccines  has  found  a  permanent  place  in  veter- 
inary treatment. 

Bacteriologic  Diagnosis. — A  mount  of  pus  stained  by  Gram's 
method  reveals  the  organism  distinctly,  if  present.  Its  character- 
istic morphology  will,  in  general,  render  its  recognition  easy. 
This  method  has  proved  to  be  of  particular  value  in  differentiating 


196  VETERINARY    BACTERIOLOGY 

this  organism  from  certain  other  pyogenic  forms,  such  as  the  gono- 
coccus.  Frequently  a  stained  mount  will  reveal  but  a  few  bacteria, 
and  cultures  are  then  necessary.  These  must  be  made  in  any 
event  where  it  is  desired  to  make  a  positive  diagnosis. 

Transmission  and  Prophylaxis. — The  fact  that  the  organism  is 
constantly  present  on  the  skin  and  hair  makes  it  particularly 
difficult  to  prevent  its  entrance  into  wounds.  The  common 
practices  of  aseptic  surgery  are  the  best  preventives  of  infection. 

Micrococcus  albus 

Synonyms. — Micrococcus  pyogenes  albus;  Staphylococcus  pyog- 
enes  albus. 

This  organism  differs  from  Micrococcus  aureus  by  being  devoid 
of  color,  and  by  being  less  pathogenic.  In  a  few  cases  of  suppura- 
tion it  has  been  found  alone,  and  in  many  cases  associated  with  the 
preceding.  In  all  other  respects  what  has  been  said  with  respect 
to  Micrococcus  aureus  will  apply  to  this  form.  It  is  believed  by 
some  investigators  that  these  two  organisms  are  simply  varieties 
of  one  single  form  which  they  term  Micrococcus  (Staphylococcus) 
pyogenes. 

Micrococcus  citreus 

Synonyms. — Micrococcus  pyogenes  citreus;  Staphylococcus  pyog- 
enes citreus;  Staphylococcus  citreus. 

This  organism  was  originally  described  by  Rosenbach  as  pres- 
ent on  the  skin.  It  is  doubtfully  pathogenic  and  relatively  un- 
common. It  differs  principally  from  Micrococcus  aureus  in  the 
production  of  a  lemon-yellow  pigment  on  agar  and  potatoes  and 
its  lack  of  power  to  liquefy  gelatin.  In  other  respects  it  resembles 
the  two  preceding  forms,  and  is  possibly  but 'a  variety  of  them. 

Micrococci  of  Uncertain  Significance 

Micrococcus  (pyogenes)  bovis  (Synonym,  Staphylococcus 
pyogenes  bovis). — This  organism  is  somewhat  smaller  than 
Micrococcus  aureus,  and  does  not  liquefy  gelatin.  It  has  been 
claimed  to  be  more  commonly  the  cause  of  suppuration  in  cattle 
than  the  typical  Micrococcus  aureus.  It  is  doubtful  whether 
there  is  a  valid  specific  difference  between  the  two. 

Micrococcus  mastitidis. — A  micrococcus  was  isolated  by  Kitt 


NON-SPECIFIC    PYOGENIC   COCCI  197 

from  mastitis  in  cows,  which  differed  from  typical  Micrococcus 
albus  principally  in  its  lack  of  power  to  liquefy  gelatin.  It  prob- 
ably represents  a  variety  merely. 

Micrococcus  ovis. — An  organism  resembling  Micrococcus  albus 
was  described  by  Nocard  as  the  cause  of  gangrenous  mastitis  in 
sheep.  With  the  exception  of  its  specialized  pathogenesis,  it 
differs  but  little  from  typical  Micrococcus  albus. 

Micrococcus  epidermidis  albus. — This  is  probably  a  variety 
of  the  Micrococcus  albus  found  in  the  deeper  layers  of  the  skin,  and 
the  common  cause  of  "  stitch  abscesses." 

Micrococcus  cereus  (albus  and  flavus). — These  organisms 
differ  from  the  micrococci  heretofore  described  in  producing  a 
waxy  growth  upon  artificial  media,  hence  the  name  cereus  (wax). 

Streptococcus  pyogenes 

Synonyms. — Streptococcus  erysipelatos ;  Str.  puerperalis;  Str. 
articulorum;  Str.  pyogenes  malignis;  Str.  septicus;  Str.  scarlatinosus. 

Pasteur  first  recognized  the  Streptococcus  in  pus,  but  Ogsten, 
between  1880  and  1884,  first  definitely  isolated  and  described  it. 
Fehleisen,  in  1883,  found  the  organism  in  erysipelas,  and  Rosen- 
bach  described  it  in  detail  and  gave  it  its  present  name. 

The  student  will  find  no  more  puzzling  group  of  organisms  than 
the  Streptococci.  They  have  been  found  in  connection  with  all 
types  of  inflammatory  processes.  Some  strains  are  exceedingly 
virulent,  others  wholly  lack  the  power  of  disease  production. 
Differences  have  been  recorded  in  the  cultural  characters  of  isola- 
tions from  different  sources,  and  the  species  split  into  several  on 
the  basis  of  these  variations.  None  of  these  classifications  has 
proved  to  be  wholly  satisfactory,  and,  for  the  present,  it  is  probably 
best  to  treat  all  the  forms  as  varieties  merely  of  one  rather  poly- 
morphic species. 

Distribution. — Streptococcus  pyogenes  does  not  adapt  itself 
as  readily  to  a  saprophytic  mode  of  existence  as  do  the  staphylo- 
cocci.  It  is  commonly  present  upon  the  skin  of  man  and  animals, 
and  has  been  isolated  from  a  great  number  of  different  inflamma- 
tory and  suppurative  processes  in  both. 

Morphology   and   Staining   Characters. — This   organism   is   a 


198  VETERINARY   BACTERIOLOGY 

coccus  about  1  ^  in  diameter,  occurring  in  chains  of  greater  or  less 
length.  Sometimes  there  is  a  tendency  toward  diplococcus 
formation,  and  the  threads  may  be  made  up  of  many  such  diplo- 
cocci.  Where  two  organisms  approximate,  they  are  more  or  less 
flattened.  The  organism  is  non-motile,  does  not  produce  spores, 
is  easily  stained  by  the  common  anilin  dyes,  and  is  gram-positive. 
The  length  and  character  of  the  chains  is  variable  in  different 
media  and  under  varying  growth  conditions,  and  likewise  in  strains 
isolated  from  different  sources.  This  latter  fact  has  been  made  use 
of  by  some  investigators  in  an  effort  to  perfect  a  classification. 


Fig.  85. — Streptococcus  pyogenes  in  lactose  broth  (Heinemann  in  "  Journal  of 
Infectious  Diseases"). 


The  typical  form  does  not  produce  capsules,  although  capsulated 
varieties  have  been  described. 

Isolation  and  Culture. — The  organism  may  frequently  be 
isolated  in  pure  culture  directly  from  the  wound,  but  it  is 
generally  necessary  to  pour  plates  and  isolate  from  the  colonies. 
Care  must  be  used  in  this  latter  method  not  to  overlook  the 
colonies,  for  many  strains  produce  minute  colonies  only,  and 
in  mixed  infections  they  may  be  missed.  Inasmuch  as  most 
strains  ferment  lactose,  with  the  production  of  acid,  the  use  of 


NON-SPECIFIC    PYOGENIC   COCCI 


199 


litmus-lactose-agar  plates  is  sometimes  helpful,  the  colony  appear- 
ing surrounded  by  a  zone  of  red. 

Growth  occurs  in  most  of  the  laboratory  media,  particularly 
upon  the  addition  of  a  sugar,  such  as  dextrose.  The  colonies 
upon  agar  and  gelatin  are  small, — rarely  larger  than  a  pinhead, 
— at  first  transparent,  and  almost  dew-drop-like.  Later  they 
may  become  somewhat  opaque.  The  gelatin  is  not  usually  lique- 
fied, although  saprophytic  strains  are  known  which  possess  this 
property.  Whether  these  latter  are  typical 
Streptococcus  pyogenes  is  uncertain.  Upon 
agar  slants  this  organism  tends  to  grow  in 
the  form  of  discrete  colonies.  Bouillon  is 
sometimes  clouded  by  a  uniform  distribu- 
tion of  short  chains;  in  other  cases  it 
remains  clear,  the  organism  growing  in 
masses  of  long,  tangled  threads  which 
remain  as  a  sediment  at  the  bottom. 
Blood-serum  is  unusually  favorable  as  a 
medium.  A  growth  is  frequently  produced 
upon  the  potato,  though  many  strains 
refuse  to  develop  on  this  medium.  Milk 
is  usually  coagulated,  with  acid  production 
and  no  digestion  of  the  curd. 

Physiology. — Streptococcus  pyogenes  is 
aerobic  and  facultative  anaerobic.  Its 
optimum  temperature  is  about  37°,  but 

growth  will    usually  take   place   at    room- 

*         rpn        xv.  i      j     xv        •  Fig.  86. — Streptococ- 

temperatures.       1  ne    thermal     deatn-point     cus  pyogenes  on  a  slant 

(Frankel 


differs  in  various  strains— usually  about  60° 
for  fifteen  minutes  is  sufficient  to  destroy. 
Antiseptics  and  disinfectants  are  efficient  in  destruction.  No 
pigment  is  produced.  No  coagulating  or  proteolytic  enzymes  are 
developed  in  the  typical  strains,  although,  as  indicated  above, 
gelatinase  has  been  reported  in  some  saprophytic  types.  No 
indol  is  produced.  No  gas  is  developed  in  any  medium.  Acid 
is  produced  by  most  strains  from  many  sugars,  particularly 
dextrose  and  lactose.  Efforts  have  been  made  to  classify  the 
various  types  of  Streptococcus  on  the  basis  of  their  acid-production 


200  VETERINARY   BACTERIOLOGY 

in  various  sugars.  This  seems  to  be  helpful  in  determining  the 
origin  of  intestinal  types  in  some  cases. 

Pathogenesis. — Unexplained  differences  and  variations  in 
pathogenesis  may  be  noted  in  the  various  strains  which  have 
been  studied.  Virulence  for  a  given  species  of  animal  may  be 
increased  by  passage  through  that  species. 

Mechanism  of  Disease  Production. — Little  more  is  known  of  the 
mechanism  of  disease  production  with  this  organism  than  with  the 
staphylococci.  Neither  the  hemolytic  streptolysin  nor  the  endo- 
toxins,  which  have  been  described,  seem  adequately  to  explain  its 
pathogenesis.  Changes  are  usually  brought  about  in  tissues 
which  are  in  more  or  less  intimate  contact  with  the  organism. 
No  class  of  infections  shows  better  the  necessity  of  virulence  of 
an  organism  and  lack  of  resistance  on  the  part  of  the  tissues  in 
order  to  bring  about  pathologic  changes. 

Experimental  Evidence  of  Pathogenesis. — Inoculation  experi- 
ments of  animals  have  duplicated  practically  every  infection  with 
which  this  organism  has  been  found  associated  in  man  and 
animals.  The  causal  relationship  of  the  organism  to  many  in- 
fections has  been  abundantly  demonstrated.  All  laboratory 
animals  may  be  infected  with  strains  exhibiting  sufficient 
virulence. 

Disease  and  Lesions  Produced. — Streptococcus  pyogenes  is  asso- 
ciated as  primary  cause  with  a  long  list  of  affections  in  both  man 
and  animals.  As  a  secondary  invader  it  is  of  great  importance 
in  many  other  diseases.  Mixed  infections  with  Micrococcus 
aureus  and  other  organisms  are  common.  Some  of  the  more 
important  are  worthy  of  note. 

Wound  Infection  and  Suppuration. — Streptococcus  pyogenes  is 
not  as  common  in  surgical  wounds  and  other  traumata  as  the 
Micrococcus  aureus  and  M.  albus.  Karlinski,  in  1890,  examined 
suppurative  processes  in  man,  animals,  and  birds,  and  found 
that  Streptococcus  was  present  in  about  22  per  cent,  of  the  cases 
in  man,  27  per  cent,  in  lower  animals,  and  15  per  cent,  in  birds. 
Staphylococci  were  present  in  about  70  per  cent.,  54  per  cent., 
and  55  per  cent,  respectively.  Lucet  found  Streptococci  alone 
in  9  cases,  and  associated  with  other  organisms  in  10  cases,  out  of 
a  total  of  52  examinations  of  abscesses  in  cattle.  Both  Strcplo- 


NON-SPECIFIC   PYOGENIC    COCCI  201 

cocci  and  Micrococci  are  commonly  present  in  fistulas  and  in  poll- 
evil  in  horses. 

Septicemia  and  Pyemia. — When  an  unusually  virulent  Strepto- 
coccus gains  entrance  to  the  blood-stream  it  may  cause  septicemia 
(blood-poisoning).  Direct  growth  through  a  blood-vessel  wall 
may  result  in  the  formation  of  an  infected  blood-clot,  and  later, 
when  broken  up,  it  produces  thrombi  and  may  lead  to  embolism. 
Abscess  formation  proceeds  at  the  new  foci  of  infection.  Multiple 
metastatic  abscess  formation  of  this  character  is  known  as  pyemia. 

Erysipelas. — This  infection  in  man  is  characterized  by  a  severe 
inflammation  of  the  skin,  in  which  this  organism  is  present  in 
large  numbers  in  the  lymph-spaces  of  the  subcutaneous  tissue. 
Erysipelas  seems  to  be  due  to  an  invasion  with  a  peculiarly  patho- 
genic organism  and  to  a  lack  of  resistance  on  the  part  of  these 
tissues.  Somewhat  similar  lesions  have  been  noted  upon  lower 
animals.  The  erysipelas  of  swine-  must  not  be  confused  with  this 
disease,  as  it  is  caused  by  a  totally  different  organism. 

Infection  of  Mucous  Surfaces. — Tonsillitis  in  man,  enteritis 
in  children,  non-diphtheritic  anginas,  and  similar  inflammations 
of  mucous  surfaces  are  commonly  caused  by  Streptococcus. 
Puerperal  fever,  an  infection  of  a  mucous  surface  following  child- 
birth and  its  consequent  septicemia,  has  been  demonstrated  in 
many  cases  to  be  due  to  Streptococcus  infection,  not  infrequently 
contracted  from  a  case  of  erysipelas.  Less  is  known  of  the  rela- 
tionship of  Streptococcus  to  related  diseases  in  animals,  though 
doubtless  it  plays  an  important  part. 

Peritonitis  following  an  enterotomy  is  usually  due  to  infection 
of  the  peritoneum  with  Streptococcus  pyogenes,  although  other 
organisms  are  equally  capable  of  giving  rise  to  this  condition. 

Pneumonia,  particularly  the  traumatic  pneumonia  of  the  horse, 
may  be  caused  by  this  organism.  The  so-called  contagious 
pleuropneumonia  of  the  horse  is  believed  by  many  investigators 
to  be  due  to  an  organism  which  has  not  been  shown  to  differ 
materially  from  Streptococcus  pyogenes,  except  for  its  exceptional 
virulence  and  mode  of  attack.  The  disease  is  contagious,  and  in 
this  respect  differs  from  most  types  of  streptococcic  infection. 

Ulcerative  Endocarditis. — This  affection  is  produced  most 
commonly  by  Streptococcus,  although  Micrococcus  is  found  in 


202  VETERINARY  BACTERIOLOGY 

some  cases.  Cauliflower-like  excrescences  of  the  heart-valves, 
with  consequent  valvular  insufficiency  and  embolism,  due  to  the 
breaking  off  of  these  particles,  are  the  characteristic  lesions. 

Arthritis. — Inflammation  of  the  bone  covering  (periostitis), 
infections  of  the  bone-marrow  (osteomyelitis),  and  of  the  joints 
(arthritis)  are  generally  the  result  of  streptococcic  invasion. 
In  all  new-born  animals  there  is  danger  of  infection  through  the 
navel,  and  consequent  production  of  "  navel  ill  "  (omphalophlebi- 
tis).  The  umbilical  vein,  according  to  Moore,  becomes  heavily 
infected  with  bacteria,  which  invade  the  joints  by  metastasis. 
The  selective  action  of  the  organisms  in  infecting  certain  tissues 
at  one  time  and  others  at  another  is  not  well  understood.  Rheu- 
matic fever  in  man  (acute  articular),  and  probably  in  animals, 
has  been  shown  to  be  sometimes  due  to  Streptococcus  pyogenes. 
The  infection  atrium  in  man  appears  to  be  the  tonsils. 

Suppurative  Cellulitis. — Under  this  heading  Moore  has  described 
the  streptococcic  infections  of  the  subcutaneous  tissues,  par- 
ticularly of  the  lower  extremities.  In  sheep  it  is  called  locally 
"  foot-rot." 

Mastitis. — Mastitis  is  commonly  caused  by  Streptococcus. 
Here  again  the  particular  strain  selects  a  particular  organ,  and 
may  be  transferred  from  one  animal  to  another  by  the  hands  of 
the  milker.  Several  different  species  of  the  Streptococcus  have 
been  described  as  associated  with  garget  or  infectious  mastitis, 
but  there  does  not  seem  to  be  any  good  reason  for  believing  them 
to  be  anything  but  specialized  strains  of  the  Streptococcus  pyogenes. 
As  will  be  seen  later,  the  presence  of  Streptococcus  in  milk  does 
not  necessarily  predicate  mastitis,  for  non-pathogenic  forms  are 
common. 

Immunity. — A  hemolytic  toxin,  streptolysin,  may  be  demon- 
strated in  some  strains  of  Streptococcus  pyogenes.  For  this  an 
antitoxin  has  been  produced.  It  seems  probable  that  there  is 
also  a  toxin  which  injures  or  destroys  leukocytes.  These  toxins 
vary  in  amount,  however,  in  cultures  of  different  strains,  and  seem 
to  vary  independently  of  the  virulence.  They  certainly  do  not 
account  for  the  pathogenic  nature  of  the  organism.  Agglutinins 
may  occasionally  be  demonstrated,  but  are  not  of  diagnostic 
importance.  Endotoxins  are  produced  by  both  virulent  and  non- 


NON-SPECIFIC   PYOGENIC   COCCI 


203 


virulent  strains.  Bacteriolysins  are  probably  not  important. 
Opsonins,  normal  and  immune,  and  the  phagocytosis  induced  by 
them,  probably  explain  any  immunity  which  is  exhibited  by  the 
body.  This  immunity,  like  others  produced  by  opsonins,  is  not 
lasting;  in  fact,  it  is  so  transient  that  it  may  be  said  that  an  attack 
of  erysipelas,  for  example,  renders  one  even  more  subject  to  recur- 
rence. 

Treatment  by  the  use  of  bacterins,  and  particularly  autogenic 
vaccines,  has  been  found  successful  in  chronic  suppurations  in 
both  man  and  animals.  In  acute  attacks  it  is  of  little  or  no  value. 
It  seems  to  be  the  consensus  of  opinion  among  investigators  that 
the  use  of  a  polyvalent 
vaccine  is  more  efficaci- 
ous than  a  univalent 
when  it  is  not  autogenic. 

Antistreptococcic  sera 
are  produced  for.  use  by 
both  the  veterinarian 
and  the  physician.  The 
serum  of  Marmorek  is 
prepared  by  the  use  of 
a  culture  having  such 
virulence  for  rabbits 
that  j^ooo  c-c-  proves 
fatal.  Horses  are  im- 


Fig. 


87. — Streptococcus     pyogenes 
(Frankel  and  Pfeiffer). 


in     pus 


munized  by  repeated  in- 
jections  of    such    broth 

cultures  and  their  serum  used.  This  seems  to  be  of  distinct 
benefit  in  immunizing  passively  an  animal  against  the  same 
strain,  but  does  not  protect  against  others.  Polyvalent  sera 
are  prepared  by  immunizing  horses  against  a  considerable  number 
of  strains  of  the  Streptococcus.  The  results  from  the  use  of 
such  sera  have  not  proved  as  successful  as  hoped,  though  some 
have  reported  excellent  results.  Such  a  serum  doubtless  owes 
its  protective  influence  to  its  opsonic  content,  but  Hektoen  and 
Ruediger  have  shown  that  in  some  antistreptococcic  sera  on  the 
market  the  opsonin  content  was  below  normal.  The  advisability 
of  the  use  of  antistreptococcic  serum  in  general  streptococcic 


204  VETERINARY    BACTERIOLOGY 

infections  in  veterinary  practice  cannot  be  determined  at  present 
from  the  data  available.  It  is  possible  that  in  some  diseases, 
particularly  of  equines,  a  serum,  either  curative  or  prophylactic, 
prepared  from  a  homologous  organism,  may  prove  practicable 
and  helpful. 

Diagnosis. — Smears  of  pus  stained  with  methylene-blue  or 
by  Gram's  method  will  usually  reveal  the  organism  if  present 
in  any  numbers.  Sometimes  the  pus  seems  to  be  practically 
sterile  upon  microscopic  examination,  but  cultures  prepared  from 
it  will  reveal  the  presence  of  the  Streptococcus. 

Transmission  and  Prophylaxis. — The  constant  presence  of 
various  strains  of  this  organism  upon  the  hair  and  skin  makes  the 
prevention  of  its  entrance  into  wounds  difficult.  Cleanliness 
and  the  use  of  mild  antiseptics  are  the  only  practicable  methods 
of  inhibiting  its  growth.  In  those  infections  which  are  character- 
ized by  a  specialized  organism,  and  which  are  more  or  less  con- 
tagious, isolation  of  infected  animals  and  quarantine  of  those 
exposed,  with  disinfection  of  barns,  particularly  stalls  and  mangers, 
are  the  methods  which  must  be  used  in  checking  the  spread. 

Streptococcus  lacticus 

Synonyms. — Bacillus  lactici  acidi. 

Strangely  enough,  the  organisms  first  isolated  from  milk,  and 
described  as  the  cause  of  its  souring,  are  not  the  ones  now  re- 
garded as  most  commonly  associated  with  this  change.  These 
were  members  of  the  intestinal  group,  or  bacilli  that  produce  both 
acid  and  gas  in  milk.  Leishman,  in  1899,  gave  the  name  Bacillus 
lactici  acidi  to  an  organism  which  he  isolated  from  soured  milk, 
and  regarded  as  the  common  cause  of  this  fermentation.  Kruse 
later  showed  that  Leishman  had  been  mistaken  in  regarding  it 
as  a  bacillus,  and  renamed  the  organism  Streptococcus  lacticus. 
Since  then  experimental  data  have  accumulated,  which  seem  to 
demonstrate  quite  conclusively  that  normal  souring  of  milk  is 
brought  about  in  the  vast  majority  of  cases  by  this  organism. 
The  work  of  Heinemann  in  this  country  served  materially  to  clear 
up  the  relationship  between  this  and  other  forms. 

Distribution. — Streptococcus  lacticus  is  found  generally  in  milk, 
butter,  and  cheese,  upon  the  skin  and  hair  of  cattle,  and  in  feces. 


NON-SPECIFIC   PYOGENIC   COCCI  205 

Isolation,  Morphology,  and  Culture. — This  organism  may  be 
most  easily  isolated  from  milk  by  plating  in  litmus-lactose  gelatin. 
The  characteristic  non-liquefying  colonies  surrounded  by  red 
may  be  easily  recognized.  Its  morphologic  characters  differ 
in  no  marked  degree  from  Streptococcus  pyogenes.  Culturally, 
many  strains  of  this  latter  organism  appear  to  be  identical  with 
Str.  lacticus.  In  fact,  the  resemblance  is  so  close  that  there  is  a 
good  reason  to  believe  that  Str.  lacticus  is  a  strain  of  Str.  pyogenes 
which  has  adapted  itself  to  a  saprophytic  life. 


Fig.  88. — Streptococcus  lacticus  (Heinemann,  in  "  Journal  of  Infectious 

Diseases"). 


Physiology. — Its  physiologic  characters  differ  in  no  noteworthy 
degree  from  the  preceding.  Acid  is  produced  from  dextrose  and 
lactose.  According  to  Heinemann,  the  dextro  acid  is  produced 
by  this  organism  and  the  levo  by  the  B.  lactis  acrogenes  and  other 
members  of  the  intestinal  group. 

The  heat  resistance  of  Str.  lacticus  is  of  particular  importance 
in  connection  with  pasteurization  of  milk.  It  has  been  generally 
regarded  that  pasteurization  would  certainly  kill  all  organisms 
of  this  type,  and  it  has  been  believed  that  pasteurized  milk  would 
contain  only  the  spores  of  putrefactive  bacteria,  and  if  improperly 


206  VETERINARY   BACTERIOLOGY 

handled,  would  "  rot."  Ayers  and  Johnson  have  shown  recently 
the  falsity  of  this  assumption.  There  are  practically  always 
present  in  any  given  sample  of  raw  milk  one  or  more  strains  of 
the  lactic-acid  organism,  which  will  resist  the  temperature  of  the 
pasteurizer,  and  will  bring  about  normal  souring  in  the  milk 
pasteurized. 

The  production  of  lactic  acid  in  milk  is  of  considerable  im- 
portance in  preventing  the  growth  of  undesirable  bacteria.  Milk 
entirely  freed  from  these  forms,  as  by  heating  to  the  boiling-point, 
does  not  sour,  but  undergoes  a  putrefactive  fermentation,  brought 
about  by  organisms  ordinarily  held  in  abeyance  by  the  develop- 
ment of  the  lactic  acid. 

Pathogenesis. — Heinemann,  by  a  series  of  animal  inoculations, 
has  shown  it  possible  to  exalt  the  virulence  of  typical  Str.  factious 
so  that  it  would  kill  rabbits  as  quickly  as  virulent  Str.  pyogenes. 

The  close  relationship  of  the  two  forms  can  scarcely  be  ques- 
tioned. Before  the  work  of  Heinemann  it  had  been  concluded 
by  many  workers  that  the  presence  of  streptococci  in  milk  was 
necessarily  an  indication  of  udder  infection,  and  examination  for 
streptococci  was  made  a  part  of  the  routine  work  of  certain 
board  of  health  laboratories.  Inasmuch  as  it  is  now  known  that 
the  Str.  lacticus  is  normally  present  in  practically  all  milk,  it  is 
evident  that  such  examinations  are  of  little  use.  A  streptococcic 
milk  standard  is  not  practicable. 

Utilization. — Different  strains  of  Streptococcus  lacticus  are 
used  in  pure  cultures  as  starters  in  the  dairy.  The  flavor  of  butter 
is  largely  dependent  upon  the  development  of  a  peculiar  flavor 
and  aroma,  largely  through  the  agency  of  the  lactic-acid  bacteria 
present.  It  is  customary,  therefore,  to  pasteurize  cream  to  destroy 
undesirable  organisms  and  to  add  to  it  the  starter,  which  is  allowed 
to  increase  and  produce  the  desired  aroma  and  flavor.  The 
Str.  lacticus  is  likewise  of  importance  in  the  manufacture  of  cheese; 
the  change  of  the  lactose  present  to  lactic  acid  is  an  essential 
preliminary  in  most  cases  to  the  ripening  process. 


CHAPTER  XXII 

SPECIFIC  INFECTIOUS  DISEASES   PRODUCED  BY  COCCI 

No  hard-and-fast  lines  can  be  drawn  between  the  specific 
and  non-specific  infections  produced  by  cocci.  The  relationship 
and  possible  identity  of  some  of  the  organisms  here  discussed,  with 
the  forms  discussed  in  the  preceding  chapter,  will  be  made  evident. 
Some  of  the  infections  here  classed  as  specific  should  possibly  be 
considered  under  the  general  heading  of  Streptococcus  pyogenes. 
The  diseases  described  are,  however,  recognized  as  clinical  entities, 
and  are,  therefore,  worthy  of  somewhat  more  lengthy  consideration. 

Several  of  the  diseases  here  discussed  are  caused  by  Strepto- 
cocci: strangles  in  horses  (Streptococcus  equi),  apoplectiform 
septicemia  in  chickens  (Str.  gallinarum),  verrucose  vaginitis  of 
cattle  (Str.  sp.),  abortion  in  mares  (Str.  sp.);  the  remainder  are 
caused  by  Micrococci:  pneumonia  (Micrococcus  pneumonia  or 
lanceolatus) ,  epidemic  cerebrospinal  meningitis  in  man  (M.  menin- 
gitidis)  and  in  horses  (M.  intracellularis  equi) ,  Malta  fever  in  goats 
and  man  (M.  melitensis) ,  takosis  in  goats  (M.  caprinus) ,  gonorrhea 
in  man  (M.  gonorrhoea),  and  botryomycosis  in  various  domestic 
animals  (M.  ascoformans) . 

The  Streptococci  of  this  group  all  closely  resemble  the  Strepto- 
coccus pyogenes,  and  possibly  are  specialized  varieties  of  that  organ- 
ism. Many  of  the  Micrococci  are  characteristically  grouped  as 
diplococci,  particularly  in  the  tissues. 

Streptococcus  equi 

Synonyms. — Streptococcus  coryzce  contagiosce  equorum. 

Disease  Produced. — Strangles  or  distemper  in  equines. 

The  disease  strangles  in  horses  has  a  long  recorded  history. 
It  was  described  by  Solleysel  in  1664,  and  its  contagious  nature 
determined  by  Lafosse  in  1790.  Schiitz  (1888)  first  isolated  and 

207 


208  VETERINARY    BACTERIOLOGY 

described  the  organism  now  generally  considered  to  be  the  specific 
cause. 

Distribution. — The  disease  is  quite  widely  distributed,  both 
in  Europe  and  America.  It  generally  attacks  young  animals. 

Morphology  and  Staining  Characters. — The  organism  is  a  gram- 
positive  Streptococcus,  resembling  the  Sir.  pyogenes  in  both  mor- 
phologic and  staining  characters.  Some  authors  state  that  the 
organism  is  gram-negative,  for  it  is  decolorized  if  the  alcohol 
remains  too  long  in  contact.  The  chains  are  usually  long  and 
twisted. 

Isolation  and  Cultural  Characters. — The  organism  may  be 
isolated  in  pure  culture,  in  most  cases  from  the  deeper  portion  of 
the  characteristic  abscesses,  by  the  same  methods  used  for  Sir. 
pyogenes.  No  specific  differences  in  cultural  characters  have  been 
shown.  Bureschello  claims  that  in  many  cases  strangles  is  a 
mixed  infection  of  Staphylococcus  aureus  and  Streptococcus  equi. 
In  such  cases  plating  would  be  necessary  to  differentiate  the  species 
of  organisms  present. 

Physiology. — A  temperature  of  60°  will  destroy  the  organism 
in  an  hour,  and  80°  in  thirty  minutes.  It  is  destroyed  readily 
by  direct  sunlight  and  by  disinfectants.  It  grows  well  at  room- 
temperature,  although  its  optimum  is  blood-heat. 

Pathogenesis. — Enough  has  already  been  said  to  make  doubt- 
ful the  standing  of  strangles  as  a  specific  disease  caused  by  a 
specific  organism. 

Mechanism  of  Disease  Production. — Little  is  known  of  the 
factors  that  determine  the  pathogenicity  of  the  organism.  Prob- 
ably they  differ  little,  if  at  all,  from  those  of  Str.  pyogenes. 

Experimental  Evidence  of  Pathogenesis. — Pus  from  abscesses 
in  strangles  kills  white  mice,  with  evidence  of  acute  septicemia 
in  two  to  four  days.  An  abscess  generally  develops  at  the  point 
of  inoculation.  Rabbits  and  guinea-pigs  succumb  to  the  injection 
of  large  quantities  of  the  organism,  but  are  not  readily  infected. 
Other  animals  are  very  resistant.  Subcutaneous  inoculations  of 
the  horse  will  usually  provoke  abscess  formation  at  the  point  of 
inoculation.  Typical  strangles  in  horses  was  caused  by  Schiitz 
by  use  of  pure  cultures.  It  seems  evident,  however,  that  there 
must  be  some  predisposing  factor  to  the  disease  in  most  cases. 


SPECIFIC    INFECTIOUS   DISEASES   PRODUCED   BY   COCCI      209 

Inoculations  upon  the  nasal  mucous  membrane  frequently  fail 
to  infect.  Some  authors  are  inclined  to  believe  that  the  true  cause 
has  not  been  discovered,  and  that  Str.  equi  is  a  secondary  invader. 
Age  is  a  predisposing  factor:  young  animals  are  most  commonly 
infected.  Other  predisposing  causes  are  fatigue  and  exposure  to 
cold,  hard  work,  or  any  other  factor  that  lowers  the  vitality. 
Variations  in  virulence  wholly  unexplained  probably  account  for 
the  epidemic  character  of  the  disease. 

Disease  and  Lesions  Produced. — The  infection  atria  are  prob- 
ably the  upper  air-passages.  The  disease  may  be  produced  in 
simple  or  malignant  form.  A  catarrhal  discharge,  with  inflamma- 
tion of  the  nasal  mucous  membranes,  is  generally  first  noted,  fol- 
lowed quickly  by  a  swelling  of  the  adjacent  lymphatic  glands 
and  of  the  submaxillary  and  pharyngeal  lymph-nodes.  These 
generally  develop  into  abscesses.  The  infection  spreads  through 
the  lymph-channels,  but  generally  remains  localized  in  the  tissues 
adjacent  to  the  point  of  infection.  Metastatic  infections  may 
occur  in  practically  any  of  the  organs  of  the  body,  and  in  the 
chronic  types  of  the  disease  great  variations  in  localization  will  be 
found.  Fatal  termination  is  rare  (0  to  3  per  cent.),  but  some- 
times occurs,  due  to  septicemia,  pyemia,  or  pneumonia. 

Immunity. — Toxins  and  antitoxins,  agglutinins,  and  bacterio- 
lysins  have  not  been  satisfactorily  demonstrated  for  this  organism. 
Immunity  is  conferred  by  an  attack  of  the  disease,  but  is  transitory, 
and  the  same  animal  may  suffer  from  the  disease  a  second  time. 
Animals  over  five  years  old  are  quite  generally  immune.  Probably 
immunity  can  be  best  accounted  for  by  increased  opsonin  con- 
tent of  the  blood  and  consequent  stimulation  of  phagocytosis. 

Immunization  by  vaccination  with  living  and  with  dead  cultures 
has  not  given  wholly  satisfactory  results.  Possibly  the  injec- 
tion of  autogenic  cultures  may  have  a  protective  influence.  Todd 
has  prepared  a  vaccine  by  growing  the  organism  on  blood-serum 
for  twenty-four  hours,  then  large  flasks,  containing  10  per  cent, 
serum  broth,  are  inoculated  and  incubated  a  month.  Six  per  cent, 
of  sterile  glycerin  is  added,  and  the  material  concentrated  at 
60°  for  two  days  over  unslaked  lime.  The  organism  is  destroyed, 
and  the  material  evaporated  to  a  thick  paste.  This  is  diluted 
and  used  as  a  vaccine.  He  has  reported  favorable  results.  The 


210  VETERINARY  BACTERIOLOGY 

use  of  the  serum  of  animals  recovered  from  the  disease  and 
hyperimmunized  by  repeated  injections  has  been  followed  by 
even  better  results.  Certain  French  veterinarians  have  secured 
unusually  good  results  by  the  prophylactic  injection  of  such  a 
serum.  The  immunity  conferred  is  not  permanent.  The  cura- 
tive effect  seems  sufficient  to  warrant  its  use  in  many  cases. 

Diagnosis. — Bacteriologic  diagnosis  may  be  made  by  identi- 
fication of  a  gram-positive  Streptococcus  from  the  lesions  charac- 
teristic of  the  disease. 

Transmission. — Transmission  probably  occurs  most  frequently 
by  the  use  of  common  drinking  troughs  and  by  ingestion  of  infec- 
tive food;  more  rarely,  by  direct  contact  and  inhalation.  It  is 
probable  that  the  organism  may  remain  in  the  glands  and  folli- 
cles of  the  nasal  mucosa  after  recovery  of  some  animals,  and  such 
might  easily  infect  others. 

Streptococcus  gallinarom 

Synonyms. — Streptococcus  of  Norgaard  and  Mohler.  Strep- 
tococcus capsulatus  gallinarum  (?). 

Disease  Produced. — Apoplectiform  and  other  septicemias  in 
chickens. 

This  disease  was  first  described,  and  its  cause  isolated,  by 
Norgaard  and  Mohler  in  1902.  The  same  disease  was  studied  by 
Moore  and  Mack  in  1905.  Damman  and  Manengold  (1906) 
recorded  an  epidemic  of  "  fowl  sleeping  sickness,"  due  to  a  Strep- 
tococcus, and  later,  in  1908,  noted  a  similar  outbreak. 

Distribution. — The  disease  has  been  reported  from  the  original 
outbreak  in  Virginia,  from  northern  New  York,  from  Sweden, 
and  a  similar,  if  not  identical,  disease  from  Germany. 

Morphology. — The  organism  is  a  typical  Streptococcus  with 
chains  of  variable  length,  cells  0.6-0.8  [A  in  diameter,  stains  readily 
by  the  ordinary  anilin  dyes,  and  is  positive  to  Gram's  stain. 
With  the  exception  of  the  doubtful  difference  in  diameter,  there 
appear  to  be  no  morphologic  characters  which  differentiate  it  from 
Str.  pyogenes.  The  German  type  is  described  as  forming  capsules 
in  the  blood,  and  may  be  entirely  distinct. 

Isolation  and  Culture. — The  organism  may  be  isolated  from  the 
blood  and  internal  organs  of  affected  fowls.  It  does  not  produce 


SPECIFIC   INFECTIOUS   DISEASES   PRODUCED    BY   COCCI      211 

sufficient  acid  to  coagulate  milk,  but  differs  culturally  otherwise 
in  no  marked  degree  from  Sir.  pyogenes. 

Pathogenesis. — Experimental  #wctence.— Inoculations  of  pure 
cultures  into  fowls,  rabbits,  mice,  and  swine  are  fatal,  while  those 
into  the  guinea-pig,  sheep,  and  dog  are  not. 

Disease  and  Lesions  Produced. — The  disease  is  a  typical  sep- 
ticemia,  marked  by  parenchymatous  degenerations  and  hemor- 
rhage. Norgaard  and  Mohler  state  that  fowls  frequently  die  in 
twelve  to  twenty-four  hours  after  the  first  symptoms. 

Immunity. — Little  is  known  relative  to  the  causal  organism 
and  its  products.  Probably  they  differ  little,  if  at  all,  from  those 
of  Str.  pyogenes.  The  dis- 
coverers state  that  active 
immunity  may  be  conferred 
by  the  injection  of  bouillon 
culture  filtrates  and  by  vac- 
cination with  killed  cult- 
ures, and  passive  immunity 
by  the  injection  of  the 
blood-serum  of  an  immun- 
ized animal. 

Bacteriologic  Diagnosis. 
— The    organism    may    be 
identified  as  a  gram-positive 
organism  in  smears  from  the  Fig    sg._Streptococcus  gamnarum  (Mag- 
blood  and  internal   organs  nusson). 

and  by  culture. 

Transmission. — According  to  the  German  authorities,  the 
disease  is  not  very  contagious,  but  its  appearance  in  considerable 
numbers  of  fowls  at  one  time  remains  unexplained. 

Streptococcus  Sp. 

Synonym. — Streptococcus  of  Ostertag. 

Disease  Produced. — Contagious  granular  or  verrucose  vaginitis 
in  cattle. 

Ostertag,  in  1898,  isolated  and  cultivated  a  Streptococcus 
from  the  purulent  discharge  and  from  the  deeper  layers  of  the 
mucous  membrane  in  cases  of  infectious  vaginal  catarrh.  His 


212  VETERINARY   BACTERIOLOGY 

findings  have  been  confirmed  by  the  work  of  Hecker,  Raebiger, 
and  Hess.  The  latter  edited  a  report  of  the  Swiss  Veterinary 
Surgeons  Society  in  1903,  which  gives  a  very  complete  summary 
of  the  knowledge  of  the  disease  and  its  cause. 

Distribution. — The  disease  has  been  reported  from  all  parts 
of  Europe,  and  is  wide-spread  in  North  America. 

Morphology. — The  organism  occurs  in  short  chains  of  six  to 
nine  individuals,  held  together  by  a  delicate  capsule.  It  stains 
readily  with  common  anilin  dyes,  and  is  decolorized  by  Gram's 
method.  This  latter  differentiates  it  sharply  from  the  Sir.  pyog- 
enes. 

Isolation  and  Culture. — Isolation  may  be  made  upon  agar  or 
gelatin.  Plate  cultures  are  usually  necessary,  as  there  are  gener- 
ally many  other  bacteria  constantly  present  in  the  infected  vagina. 
Growth  occurs  on  gelatin  (without  liquefaction),  blood-serum, 
and  agar,  particularly  glycerinized.  It  produces  a  diffuse  cloud- 
ing of  bouillon.  Acid  production  is  so  weak  that  milk  is  not 
coagulated.  It  will  be  noted  that  the  latter  is  also  a  character 
which  differentiates  it  from  the  Str.  pyogenes. 

Physiology. — The  organism  is  aerobic  and  facultative  anaerobic. 
Acids  are  produced  in  small  quantities,  if  at  all,  in  carbohydrate 
media.  The  optimum  growth  temperature  is  blood-heat,  but 
good  growth  occurs  at  room-temperature. 

Pathogenesis. — Experimental  Evidence. — This  organism  is  not 
pathogenic  for  any  of  the  laboratory  animals,  nor  can  it  produce 
disease  in  horses,  hogs,  sheep,  or  dogs  when  inoculated  into  the 
vagina.  Inoculations  of  pure  cultures  into  the  vagina  of  heifers 
has  been  found  to  reproduce  the  disease,  so  that  there  seems  to  be 
little  doubt  of  its  etiologic  relation  to  the  disease.  It  seems  to  be  an 
example  of  extreme  specialization,  such  as  is  found  in  the  organism 
causing  gonorrhea  in  man. 

Disease  and  Lesions  Produced. — The  disease  in  an  acute  form 
produces  a  swelling  of  the  labia  of  the  vulva,  with  increased  secre- 
tion from  the  mucous  membranes  of  the  vagina.  Later  the  dis- 
charge becomes  purulent,  then  granules  from  the  size  of  a  pin- 
head  to  a  rape-seed  develop.  These  are  the  enlarged  lymph-fol- 
licles of  the  mucous  membrane.  The  acute  stage  lasts  usually 
for  several  weeks,  the  discharge  becomes  less  prominent,  and 


SPECIFIC    INFECTIOUS   DISEASES   PRODUCED   BY   COCCI      213 

finally  the  chronic  stage  sets  in,  and  may  persist  indefinitely  or 
gradually  disappear.  The  organism  may  be  isolated  from  the 
pus  in  the  acute  stage,  or  from  the  deeper  layers  of  the  mucosa 
in  the  later  stages.  In  the  bull  the  glans  penis  may  show  gran- 
ules similar  to  those  of  the  vagina,  or  there  may  be  a  purulent 
catarrh  of  the  prepuce.  In  the  cow  the  disease  may  involve  the 
uterus  and  possibly  produce  abortion  and  even  sterility. 

Immunity. — Recovery  from  the  disease  presumably  results 
from  the  development  of  an  active  immunity,  probably  opsonic 
in  nature.  No  means  of  artificial  immunization  have  been 
employed. 

Bacteriologic  Diagnosis. — The  presence  of  a  gram-negative 
Streptococcus  in  the  depths  of  the  mucous  membranes  should  be 
diagnostic  of  the  disease.  Identification  in  the  discharges  would 
necessitate  cultures. 

Transmission. — The  disease  is  probably  most  commonly  trans- 
mitted by  coition  or  by  immediate  contact  with  soiled  litter  and 
fodder. 

Streptococcus  Sp. 

Disease  Produced. — Contagious  abortion  in  mares.  Ostertag 
in  1900  described  a  Streptococcus  as  the  cause  of  abortion  in  mares. 

Distribution. — Recorded  only  from  Europe. 

Morphology. — This  organism  somewhat  resembles  the  preced- 
ing morphologically.  It  is  a  coccus,  occurring  in  short  chains, 
stains  easily  with  anilin  dyes,  but  is  gram-negative. 

Isolation  and  Culture. — Ostertag  succeeded  in  isolating  the 
organism  upon  blood-serum  in  pure  cultures  from  the  uterine 
mucous  membranes  and  from  the  blood  and  internal  organs  of  an 
aborted  fetus.  This  Streptococcus  does  not  grow  readily  upon 
most  artificial  media,  but  shows  much  better  development  upon  the 
addition  of  blood-serum.  Serum  bouillon  shows  initial  clouding, 
with  subsequent  sedimentation.  Upon  the  solid  serum  media  the 
growth  is  poor,  being  scarcely  visible  to  the  naked  eye.  Milk 
is  not  changed.  It  will  be  noted  that  both  this  organism  and  the 
preceding  may  be  differentiated,  both  morphologically  and  cul- 
turally from  the  Str.  pyogenes. 

Physiology. — The  organism  quickly  dies  on  artificial  media, 


214  VETERINARY   BACTERIOLOGY 

frequent  transplantations  being  necessary  to  maintain  it.  It  is 
easily  destroyed  by  disinfectants. 

Pathogenesis. — Ostertag  succeeded  in  producing  abortion  in  a 
pregnant  mare  by  an  intravenous  injection.  The  organism  is 
not  pathogenic  to  the  common  laboratory  animals.  Like  the 
preceding,  it  seems  to  be  a  case  of  specialized  parasitism.  Not 
enough  critical  work  has  been  done  upon  the  disease  to  satis- 
factorily establish  the  causal  relationships  of  this  organism. 

Immunity. — No  work  is  recorded  relative  to  immunity  to  this 
disease.  Transmission  is  probably  through  coitus. 

Other  Streptococci  of  Uncertain  Significance 

Streptococcus  mastitidis  sporadicae. — (Str.  agalactice  conta- 
giosce,  Str.  der  infektiosen  Induration  des  Euters). 

Organisms  of  mammitis  or  mastitis  in  cattle  have  been  found 
in  all  parts  of  the  world,  frequently  associated  with  the  disease  in 
epidemic  form.  This  organism  was  originally  reported  as  gram- 
negative,  but  the  Str.  mastitidis,  gram-positive,  is  reported  by  the 
local  government  board  in  England  as  the  commoner  type.  There 
are  no  good  differential  characters  other  than  pathogenesis  to 
separate  this  latter  form  from  Str.  pyogenes  and  Str.  lacticus. 
On  account  of  the  general  occurrence  of  this  latter  species  in  milk, 
direct  microscopic  examination  of  the  milk  is  often  insufficient 
for  the  purpose  of  determining  the  condition  of  the  udder,  i.  e., 
whether  or  not  it  is  infected  with  garget. 

Streptococcus  Sp. — Epizootic  pleuropneumonia  in  equines. 
Stable  pneumonia. 

A  number  of  investigators  have  demonstrated  a  Streptococcus 
present  in  the  lesions  of  certain  pneumonias  in  the  horse.  The 
organisms  isolated  by  different  investigators  have  not  always  the 
same  cultural,  morphologic,  and  physiologic  characteristics.  By 
some  the  organism  is  regarded  as  identical  with  Str.  equi,  by 
others  as  a  specific  type,  and  by  still  others  it  is  believed  that 
the  specific  organism  is  still  unknown,  and  the  Streptococci  de- 
scribed are  but  secondary  invaders.  An  organism  resembling  the 
human  pneumococcus,  if  not  identical  with  it,  has  been  isolated 
from  some  cases  of  equine  pneumonia,  and  will  be  considered  in 
connection  with  that  organism. 


SPECIFIC   INFECTIOUS   DISEASES   PRODUCED    BY   COCCI      215 


Streptococcus  pneumonias 

Synonyms. — Pneumococcus;  Diplococcus  pneumonia;  Diplo- 
coccus lanceolatus;  Streptococcus  pneumonice;  Micrococcus  pneu- 
monice;  Micrococcus  lanceolatus. 

Disease  Produced. — Pneumonia  in  man,  and  probably  some 
animals,  particularly  the  horse. 

Sternberg,  in  1880,  described  this  organism  from  normal 
sputum,  but  Frankel,  in  1885,  first  definitely  associated  the  organ- 
ism with  croupous  pneumonia.  Considerable  difficulty  early 
arose,  due  to  the  confusion  of  two  distinct  organisms;  namely, 
the  form  under  consideration  with  the  pneumobacillus  of  Fried- 
lander.  A  somewhat  similar  organism,  possibly  a  gram-negative 
variety  of  this  form,  has  been 
found  by  Mayer  associated  with 
pneumonia  in  the  horse. 

Distribution.  — Throughout 
the  world,  in  diseased  and 
healthy  individuals. 

Morphology  and  Staining. — 
Usually  occurs  in  twos,  more 
rarely  in  chains  of  four  or  six, 
spherical  or  more  generally  flat- 
tened at  the  point  of  contact, 
and  with  opposite  side  some- 
what elongated  and  pointed, 
whence  the  name,  lanceolatus. 
Capsules  may  be  demonstrated 

in  the  body,  but  do  not  appear  in  culture-media  except  in 
serum  broth.  It  stains  readily  with  ordinary  anilin  dyes,  and  is 
gram-positive.  There  is  some  doubt  as  to  whether  the  organism 
is  a  Micrococcus  or  a  Streptococcus.  The  occurrence  of  chain- 
formation  and  lack  of  any  other  grouping  would  make  the  latter 
more  probable. 

Isolation  and  Cultural  Characters. — The  organism  may  in  some 
cases  be  isolated  from  the  blood  directly  in  pure  cultures.  It 
is  most  readily  obtained  from  the  sputum  by  animal  passage. 
Growth  occurs  on  most  laboratory  media  except  potato.  Growth 
is  never  luxuriant,  the  organism  developing  as  discrete,  transparent, 


Fig.  90. — Streptococcus  pneumonice 
in  pure  culture  (Weichselbaum)  (Kolle 
and  Wassermann). 


216 


VETERINARY   BACTERIOLOGY 


dewdrop-like  colonies  upon  the  surface  of  the  medium.  Bouillon 
is  slightly  clouded.  Milk  is  acidified  and  coagulated.  The 
addition  of  glycerin,  and  particularly  blood-serum,  stimulates 
growth  in  most  media. 

Physiology.  —  The  optimum  temperature  is  37°;  little  or  no 
growth  will  occur  at  lower  temperatures.  Desiccation  for  several 
months,  particularly  in  sputum,  does  not  always  kill  the  organ- 
ism. Twelve  hours'  exposure  to  direct  sunlight  is  fatal.  Acids 
are  produced  from  many  carbohydrates. 

Pathogenesis.  —  The  pneumococcus  produces  acute  septicemia 

when  injected  into  the 
mouse,  guinea-pig,  or  rab- 
bit. The  production  of 
typical  pneumonia  is  at- 
tended with  difficulty,  if, 
indeed,  it  has  been  satis- 
factorily demonstrated.  Its 
principal  claim  to  recogni- 
tion as  the  etiologic  factor 
in  the  disease  rests  upon 
its  presence  in  the  lesions 
and  its  general  pathogenic 

relationship  to  animals. 
Thg  f  h  .  .  ; 

commonly  present  in  the 
sputum  of  normal  individuals  seems  to  ihdicate  that  there  are 
great  differences  in  disease-producing  power  among  different 
strains.  Whether  or  not  the  organism  isolated  by  Mayer  from 
pneumonia  in  the  horse  is  identical  with  this  organism  cannot  at 
present  be  determined,  nor  can  its  relationship  to  "  Brustseuche  " 
be  said  to  be  satisfactorily  proved. 

The  tissues  of  the  lung  invaded  by  the  organism  become  con- 
gested, and  blood-plasma  is  poured  into  the  alveoli.  The  fib- 
rinogen  coagulates,  and  the  lung  becomes  "  hepatized,"  that 
is,  liver-like  in  consistency.  Frequently  there  is  more  or  less 
hemorrhage,  largely  by  diapedesis,  and  the  lung  becomes  red- 
dened. Later  leukocytes,  particularly  the  polymorphonuclear 
type,  invade,  and  the  tissues  become  gray.  Autolytic  digestion 


- 
agar  plate 


SPECIFIC    INFECTIOUS   DISEASES   PRODUCED    BY    COCCI      217 

of  the  fibrin  and  other  exudates  supervenes,  and  the  material 
passes  off  through  the  air-passages  or  is  resorbed. 

Metastatic  infections  with  the  pneumococcus  are  common 
in  man.  Inflammations  of  the  endocardium,  the  pericardium, 
the  pleura,  and  the  meninges  have  been  found  to  be  due  to 
this  organism  in  a  small  percentage  of  cases.  Otitis  media 
is  sometimes  the  initial  infection,  and  may  be  followed  by 
meningitis. 

Immunity. — No  toxin  has  been  demonstrated  in  this  organism. 
Endotoxins  may  be  demonstrated,  but  whether  they  account  for 


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Fig.  92. — Streptococcus  pneumonia.     Stained  preparations  showing  capsules 
(Buerger,  in  "Journal  of  Infectious  Diseases"). 

its  pathogenicity  is  uncertain.  Agglutination  of  the  pneumo- 
coccus occurs  with  the  blood-serum  of  an  infected  individual, 
but  not  usually  in  greater  dilutions  than  1 :  50.  Opsonins,  both 
normal  and  immune,  have  been  demonstrated,  and  likewise  there 
has  been  isolated  from  virulent  Streptococci  a  substance  that 
inhibits  phagocytosis. 

Immunity  to  pneumonia  is  transient;  relapses  frequently 
occur,  possibly  due  to  decrease  in  immunity  during  convalescence. 
Recovery  in  some  cases  seems  to  be  followed  by  increased  sus- 
ceptibility. The  immunity  developed  is  evidently  opsonic  in 


218  VETERINARY  BACTERIOLOGY 

nature.  No  practicable  method  of  immunization,  either  by  the 
use  of  vaccines  or  antisera,  has  been  developed. 

Bacteriologic  Diagnosis. — The  most  conclusive  method  of 
bacteriologic  diagnosis  is  by  isolation  and  cultivation  of  the  organ- 
ism. A  demonstration  of  the  characteristic  lanceolate,  capsulated, 
gram-positive  diplococci  is  often  diagnostic.  The  agglutination 
test  is  not  conclusive. 

The  relationship  of  this  organism  to  pleuropneumonia  and 
contagious  pneumonia  in  equines  is  uncertain,  and  further  work 
needs  to  be  done  before  the  connection  can  be  made  clear. 


Micrococcus   meningitidis 

Synonyms. — Diplococcus  intracellularis  meningitidis;  Micro- 
coccus  weichselbaumii ;  Streptococcus  meningitidis;  meningococcus. 

Disease  Produced. — Epidemic  cerebrospinal  meningitis  in 
man. 

Weichselbaum,  in  1887,  first  adequately  described  this  organism 
from  meningeal  exudate,  and  proved  its  pathogenic  nature  by 
animal  experimentation.  It  has  since  been  observed  repeatedly 
in  many  epidemics,  both  in  Europe  and  America. 

Morphology  and  Staining. — Micrococcus  meningitidis  in  stained 
smears  of  meningeal  exudate  usually  appears  as  a  diplococcus, 
or  in  groups  of  four.  In  culture-media  it  is  about  1  /w  in  diameter, 
and  is  usually  in  pairs,  rarely  in  short  chains.  The  latter  fact 
would  seem  to  indicate  that  this  organism  should  be  classed  as  a 
Streptococcus,  but  the  tetrad  formation  sometimes  seen  seems, 
on  the  contrary,  to  show  that  cell  division  may  be  in  two  planes, 
hence  the  use  of  the  generic  name,  Micrococcus.  No  capsules 
are  produced.  It  stains  readily  with  the  ordinary  anilin  dyes, 
but  is  gram-negative. 

Isolation  and  Culture. — The  organism  may  be  obtained  by  a 
lumbar  puncture  with  a  sterile  hypodermic  needle,  and  transferred 
directly  to  artificial  media.  It  is  best  to  use  a  medium  containing 
serum  for  the  first  isolation,  for  this  bacterium  frequently  does  not 
grow  well  on  artificial  media  at  the  first.  Upon  blood-serum 
at  37°  white,  viscid,  coherent  colonies  develop.  Serum  may  be 
added  to  agar  or  bouillon  and  will  be  found  to  favor  the  growth. 


SPECIFIC    INFECTIOUS   DISEASES   PRODUCED    BY   COCCI      219 

Milk  is  not  changed.  Frequent  transfers  are  necessary  to  the 
preservation  of  cultures  in  artificial  media. 

Physiology. — The  meningococcus  is  killed  almost  immediately 
by  desiccation.  In  culture-media  autolysis  rapidly  occurs,  and 
the  organism  soon  disappears.  No  acids  are  produced  in  carbo- 
hydrate media.  Proteolytic  enzymes  are  not  produced. 

Pathogenesis. — The  meningococcus  does  not  readily  infect 
common  laboratory  animals  unless  intraperitoneal  injections  of 
comparatively  large  amounts  of  the  culture  be  used.  Even  in 


Fig.  93. — Micrococcus  meningitidis  in  a  preparation  of  pus  from  a  brain  abscess. 
Note  that  the  organism  is  generally  intracellular  (Flexner). 

this  case  the  result  seems  to  arise  from  the  absorption  of  the  toxic 
products,  rather  than  from  an  invasion  of  the  tissues.  Injection 
of  pure  cultures  into  the  spinal  cavity  of  the  goat  has  been  found 
to  produce  meningitis,  and  inoculation  into  the  monkey  has 
resulted  practically  in  a  duplication  of  the  disease  as  it  occurs  in 
man.  The  relationship  of  the  organism  to  the  disease  is,  therefore, 
well  demonstrated. 

The  disease  is  an  acute  inflammation  of  the  meninges,  accom- 
panied by  a  purulent  exudate.     The  organism  sometimes,  though 


220  VETERINARY   BACTERIOLOGY 

rarely,  enters  the  blood.  Metastatic  involvement  of  the  lungs 
and  other  organs  occurs  in  a  few  cases. 

Immunity. — No  true  toxins  have  been  demonstrated;  endo- 
toxins  are  probably  produced,  and  released  through  the  autolytic 
disintegration  of  the  organism.  Agglutinins  are  developed  in 
sufficient  quantity,  so  that  the  blood  of  a  patient  frequently  ag- 
glutinates in  a  dilution  of  1 :  50.  Although  no  distinct  bacterio- 
lysins  have  been  demonstrated,  specific  amboceptors  for  the  organ- 
ism may  be  shown  to  be  present  in  the  blood  by  the  hemolytic 
absorption  of  complement  test.  Opsonins  probably  play  the 
largest  part  in  the  .development  of  immunity.  All  these  anti- 
bodies mentioned  have  been  determined  to  be  present  in  the 
serum  of  immunized  horses. 

Vaccination  against  the  disease  is  not  practised.  Flexner 
and  Jobling  have  prepared  an  immune  serum  from  the  horse  by 
the  injection  of  dead  bacteria,  followed  by  the  injection  of  living 
bacteria  and  the  products  of  their  autolytic  digestion.  The 
serum  is  injected  directly  into  the  spinal  canal  after  the  withdrawal 
of  an  equal  amount  of  the  purulent  exudate.  It  comes  in  direct 
contact,  therefore,  with  the  organisms,  and  probably  stimulates 
phagocytosis  by  its  opsonin  content.  The  use  of  this  serum  has 
proved  highly  successful;  by  its  means  mortality  has  been  mate- 
rially reduced. 

Bacteriologic  Diagnosis. — Lumbar  puncture,  with  demonstra- 
tion in  smears  of  a  gram-negative  diplococcus,  occurring  prin- 
cipally within  the  leukocytes,  constitutes  a  satisfactory  diagnosis. 
The  agglutination  test  may  be  applied,  but  is  not  used  in  practice. 

Transmission  and  Prophylaxis. — How  the  organism  gains  en- 
trance to  the  spinal  and  brain  cavities  is  not  certainly  known.  It 
is  found  in  the  early  stages  of  the  disease  upon  the  nasal  mucous 
membranes.  It  is  spread  probably  by  the  use  of  infected  hand- 
kerchiefs or  by  the  inhalation  of  infectious  droplets. 

Micrococcos  intracellularis  eqoi 

Synonym. — Diplococcus  intracellularis  equi. 

An  organism  in  no  important  particular  differing  from  the 
meningococcus  has  been  reported  by  Johne,  Ostertag,  and  others 
in  epizootic  or  cerebrospinal  meningitis  in  horses.  Ostertag 


SPECIFIC    INFECTIOUS   DISEASES   PRODUCED    BY  COCCI      221 

succeeded  in  producing  the  disease  in  horses  by  subdural  injec- 
tions of  pure  cultures.  Other  organisms  have  been  found  in  similar 
outbreaks  by  other  investigators.  Careful  study  of  the  relation- 
ship of  these  organisms  to  the  diseases  must  be  made  before  con- 
clusions as  to  their  importance  as  an  etiologic  factor  would  be 
justified.  This  epizootic  infection  should  not  be  confused  with  the 
far  more  common  type  of  so-called  meningitis  produced  by  forage 

poisoning. 

Micrococcus  mclitcnsis 

Diseases  Produced. — Malta  fever  in  the  goat  and  in  man; 
Mediterranean  fever. 

Bruce,  in  1887,  discovered  a  microorganism  in  the  spleen  of 
men  dead  from  Malta  or  Mediterranean  fever.  Since  that  time 
it  has  been  studied  carefully  by  numerous  investigators,  and  its 
relationship  to  the  disease  is  well  established. 

Distribution. — The  disease  is  known  to  occur  in  all  the  countries 
bordering  on  the  Mediterranean,  in  southern  Asia,  South  Africa, 
the  Philippines,  and  some  of  the  islands  of  the  West  Indies. 

Morphology  and  Staining. — The  organism  is  a  small '  coccus, 
about  0.4  [A  in  diameter,  usually  in  pairs,  occasionally  in  short 
chains.  Possibly  it  should  be  classed  as  a  Streptococcus.  Rarely, 
forms  longer  than  broad  may  be  observed,  especially  in  cultures 
kept  at  temperatures  below  the  optimum.  These  are  probably 
involution  forms.  The  organism  stains  well  with  ordinary  anilin 
dyes,  but  is  gram-negative. 

Isolation  and  Culture. — It  may  be  isolated  from  the  spleen 
during  life  or  after  death  in  pure  culture,  or  by  plating.  The 
individual  colonies  on  agar  in  three  days  are  small  and  dew-drop- 
like,  and  continue  to  increase  in  size  for  some  time.  There  is  no 
marked  peculiarity  of  growth  upon  any  of  the  artificial  media, 
although  the  organism  may  be  cultivated  readily  on  any  of  them. 
Milk  is  not  changed. 

Physiology. — The  M.  melitensis  does  not  produce  acid  from  any 
of  the  carbohydrates.  Desiccation  does  not  destroy  it  quickly, 
for  the  organism  has  been  found  to  remain  alive  and  virulent  when 
dried  for  a  considerable  time.  Pasteurization  is  fatal. 

Pathogenesis. — The  disease  is  a  true  bacteremia.  Inoculation 
of  pure  cultures  reproduces  the  disease  in  the  goat,  cow,  and  mon- 


222 


VETERINARY   BACTERIOLOGY 


key.  Accidental  laboratory  infections  have  proved  its  power 
of  producing  disease  in  man.  Infection  probably  usually  arises 
through  ingestion.  The  disease  is  characterized  by  its  low  mor- 
tality, its  long  duration  in  man,  and  the  accompanying  articular 
rheumatism.  It  is  a  disease  primarily  of  the  goat,  though  it  is 
possible  that  cattle  may  sometimes  harbor  and  transmit  it. 

Immunity. — No  toxins  have  been  demonstrated.  Agglutinins 
are  present  in  the  blood  in  infected  individuals,  so  that  agglutina- 
tion may  sometimes  be  secured  with  high  dilutions — occasionally 
as  high  as  1 : 6000.  This  test  is  one  of  the  readiest  methods  of 
diagnosing  the  disease. 

Bacteriologic  Diagnosis. — Diagnosis  may  be  made  by  isolation 

of  the  organism  by  a  punc- 
ture of  the  spleen,  or  by 
demonstrating  the  presence 
of  specific  agglutinins  in 
the  serum  in  dilutions  of 
1 :  50  or  greater. 

Transmission  and  Pro- 
phylaxis.— The  organism  is 
excreted  in  the  feces,  urine, 
and  milk  from  infected 
animals.  Most  cases  in 
the  human  are  acquired 
by  drinking  the  milk  of  in- 
fected animals.  The  dis- 
ease infects,  in  either  the 
acute  or  chronic  form,  so 

large  a  proportion  of  the  animals  in  some  countries  that  the  use 
of  unheated  milk  is  always  attended  with  danger.  The  disease 
has  no  foothold  at  present  in  the  United  States,  but  is  occa- 
sionally reported. 

Micrococcus   caprinus 

Disease  Produced. — Takosis,  or  wasting  disease,  of  Angora 
goats. 

Mohler  and  Washburn,  in  1903,  described  the  organism  be- 
lieved to  be  the  specific  cause  of  this  disease  of  Angora  goats. 


Fig.  94. — Micrococcus  melitensis  (X  1200) 
(Jordan) . 


SPECIFIC   INFECTIOUS   DISEASES   PRODUCED    BY   COCCI      223 

Their  published  investigations  still  remain  practically  the  only 
discussion  of  the  etiology  to  the  present  time. 

Distribution. — The  disease  is  certainly  known  only  from  the 
United  States,  where  it  has  been  reported  from  many  localities, 
particularly  in  the  northern  States.  It  is  probably  to  be  found 
in  other  countries,  and  is  believed  to  be  endemic  in  Asia  Minor. 

Morphology  and  Staining. — The  M.  caprinus  occurs  in  pairs  in 
the  blood,  and  usually  shows  the  same  grouping  in  culture-media, 
rarely  in  chains  of  three  or  four  cells.  When  in  pairs,  the  cells 
are  somewhat  compressed  longitudinally.  Rarely  in  the  blood 
they  assume  the  lancet  shape  characteristic  of  Str.  pneumonias. 
No  capsules  are  produced.  The  organism  does  not  stain  well 
with  methylene-blue,  but  heavily  with  carbol-fuchsin  and  gentian- 
violet.  It  is  gram-positive. 

Isolation  and  Culture. — The  organism  has  been  isolated  directly 
from  the  blood  upon  artificial  media.     In  broth  there  is  first 
turbidity,   followed  by   sedimentation   and 
clearing   up  of   the    medium,     tlpon  agar 
slants  a  white,  ceraceous  or  granular  mass 
of  confluent  colonies  is  produced.     Colonies 
on  agar  reach  a  diameter  of  8  to  10  mm., 
and  appear  smooth,  white,  flatly  convex, 

and  waxy.     When  first  isolated,  liquefaction         Fis- 

cus  capnnus  in  a  blood- 
of  gelatin  does  not  occur,  but  later  transfers     gmear    (Mohler    and 

liquefy  the  gelatin,  beginning  at  the  fourth     Washburn) . 
day.     Good  growth  is  obtained  upon  blood- 
serum  and  potato.      Milk  is  coagulated,  with   development  of 
acids  and  ultimate  peptonization.     Many  of  the  characters  noted 
relate  the  organism  closely  to  M.  albus. 

Physiology. — The  organism  is  aerobic  and  facultative  anae- 
robic. Blood-heat  is  the  optimum  temperature,  but  develop- 
ment takes  place  at  room-temperature  also.  Acids  are  produced 
in  many  carbohydrate  media.  No  indol  is  produced,  but  phenol 
may  be  demonstrated.  The  thermal  death-point  is  58°  for  ten 
minutes.  The  organism  is  readily  destroyed  by  desiccation  and 
light  and  by  the  action  of  disinfectants. 

Pathogenesis. — The  exact  mechanism  of  disease  production  is 
not  understood.  It  certainly  is  not  by  the  development  of  toxins. 


224  VETERINARY   BACTERIOLOGY 

Experimental  Evidence  of  Pathogenesis. — The  organisms  pro- 
duce a  fatal  disease  in  mice,  guinea-pigs,  and  sometimes  rabbits, 
but  the  white  and  brown  rat,  dog,  and  sheep  are  found  to  be 
immune.  Inoculation  into  the  goat  revealed  the  fact  that  the 
disease  could  be  produced  only  with  difficulty,  yet  enough  evi- 
dence was  secured  to  make  the  demonstration  of  the  organism 
as  the  etiologic  factor  satisfactory. 

Disease  Produced. — The  disease  produced  in  the  Angora  goat  is 
characterized  by  symptoms  of  diarrhea,  by  pneumonia,  great 
emaciation,  and  weakness.  It  is  generally  fatal.  The  visible 
mucous  membranes  are  anemic.  The  anemia  is  even  more  marked 
on  postmortem  examination.  The  lungs  show  inflammatory 
changes;  the  exterior  is  frequently  mottled,  showing  much  the 
appearance  of  pneumonia  in  process  of  resolution.  Degenerative 
changes  are  also  to  be  observed  in  the  heart  and  the  kidneys. 

Immunity. — There  is  no  evidence  of  toxin  production.  At- 
tempts at  immunization  by  injection  of  broth  nitrates  into  guinea- 
pigs  revealed  the  development  of  some  increased  resistance. 
Efforts  at  practical  immunization  were  unsuccessful.  Nothing 
is  known  of  the  agglutinins,  opsonins,  or  bactericidal  properties 
of  the  immune  serum. 

Bacteriologic  Diagnosis. — Cultures  from  the  blood,  or  stained 
mounts  from  this  source,  should  reveal  the  characteristic  organism. 

Transmission. — Methods    of    transmission    are    not    certainly 

known. 

Micrococcus  gonorrhoeas 

Synonyms. — Diplococcus  gonorrhoea,  gonococcus;  diplococcus 
of  Neisser. 

Diseases  Produced. — Gonorrhea  and  its  sequelae  in  man. 

Neisser,  in  1879,  noted  the  occurrence  of  a  characteristic  coccus 
in  gonorrheal  pus.  Bumm,  in  1885,  succeeded  in  obtaining  the 
organism  in  pure  cultures,  and  determined  by  inoculations  into 
human  subjects  the  causal  relationship  of  the  organism  to  the 
disease. 

Distribution. — Gonorrhea  is  a  common  disease  in  all  classes  of 
men,  particularly  in  civilized  countries. 

Morphology  and  Staining. — The  gonococcus  closely  resembles 
the  meningococcus.  In  stained  preparations  of  gonorrheal  pus 


SPECIFIC    INFECTIOUS   DISEASES   PRODUCED    BY   COCCI      225 

the  organisms  occur  generally  in  pairs  inside  the  polymorphonuclear 
leukocytes.  The  cells  are  usually  coffee-bean  shaped.  The  in- 
dividual cells  measure  about  1.6  by  0.8  [L.  There  is  little  or  no 
tendency  to  chain  formation,  the  cells  being  arranged  in  irregular 
masses  in  culture-media.  The  organism  stains  readily  with  the 
ordinary  anilin  dyes  and  is  gram-negative.  This  latter  fact  is 
important,  as  it  renders  differential  diagnosis  between  the  com- 
mon streptococci  and  the  gonococcus  possible. 

Isolation  and  Cultural  Characters. — The  organism  may  be 
isolated  directly  from  gonorrheal  pus,  care  being  exercised  to 
secure  pus  not  contaminated  with  organisms  from  the  skin.  Con- 
siderable difficulty  is  sometimes  experienced  in  securing  cultures. 
Usually  no  growth  will  occur 
on  ordinary  agar  or  gelatin, 
although,  when  considerable 
quantities  of  pus  are  spread 
over  the  surface,  some  colonies 
will  develop.  Wertheim's  me- 
dium, composed  of  one  part  of 
human  serum  to  two  parts  of 
nutrient  agar,  is  commonly 
found  to  be  the  one  on  which 
growth  is  most  readily  secured. 
The  organism  gradually  adapts 

itself    to    growth    on    artificial     v. 

Fig.  96. — Micrococcus  gonorrhoea   m 

media,  and  after  a  few  transfers  pus  (Giinther). 

develops  more  luxuriantly.    The 

colonies  resemble  those  of  the  Sir.  pyogenes,  being  small,  discrete, 
and  transparent. 

Physiology. — The  organism  is  aerobic.  It  is  easily  destroyed 
by  desiccation,  although  when  dried  in  pus  it  may  retain  its 
vitality  for  weeks.  It  must  be  transferred  from  one  culture- 
medium  to  another  every  two  days  if  kept  at  thermostat 
temperatures,  less  frequently  in  the  refrigerator,  to  keep  it 
alive. 

Pathogenesis. — Evidence  of  Pathogenesis. — That  the  gono- 
coccus causes  gonorrhea  is  evident  from  the  fact  of  its  universal 
occurrence  in  gonorrheal  pus,  and  from  inoculation  experiments 

15 


226  VETERINARY   BACTERIOLOGY 

upon  man.  The  laboratory  animals  do  not  contract  the  disease 
upon  inoculation. 

Character  of  Disease  and  Lesions. — The  organism  ordinarily 
causes  an  acute  urethritis  in  both  sexes.  It  may  also  produce 
gonorrheal  ophthalmia,  particularly  in  the  new-born.  The 
urethritis  may  become  chronic  and  lead  to  stricture.  Secondary 
involvement  of  the  Fallopian  tubes,  ovaries,  and  peritoneum  in 
the  female,  and  of  the  epididymis  and  bladder  in  the  male,  fre- 
quently occurs.  Metastatic  infections  of  the  joints,  causing  gon- 
orrheal rheumatism,  of  the  heart  valves,  causing  endocarditis, 
of  the  brain  and  cord,  causing  meningitis,  are  not  uncommon. 
The  organism  may  persist  for  a  long  period  in  a  dormant  state. 

Immunity. — No  true  toxins  are  produced  by  the  gonococcus, 
but  endotoxins  have  been  demonstrated,  as  have  also  specific 
agglutinins  and  precipitins.  Little  or  no  immunity  is  developed 
as  the  result  of  an  infection.  The  presence  of  specific  ambocep- 
tors  in  the  blood  has  been  shown  by  the  method  of  deviation  of 
complement.  Vaccination  with  the  autolytic  products  of  the 
organism,  and  with  cultures  killed  by  heat,  and  by  mixture  with 
strong  solutions  of  lactose  or  other  sugars,  have  been  used  with 
a  moderate  degree  of  success.  Porrey  has  prepared  an  anti- 
gonococcus  serum  from  the  rabbit  by  immunization  with  living 
cultures,  and  claims  to  have  secured  favorable  results  from  its  use. 

Micrococcos    ascoformans 

Synonyms. — Micrococcus  botryogenus;  Botryococcus  ascofor- 
mans; Botryomyces  ascoformans;  Zodglcea  pulmonis  equi. 

Disease  Produced. — Botryomycosis. 

The  organism  associated  with  botryomycosis  was  discovered 
in  1870,  and  later  investigated  independently  by  Revolta  and 
Micellone  in  1879,  by  Johne  in  1885,  and  by  Rabe  in  1886. 

Distribution. — This  infection  has  been  reported  several  times 
from  Europe,  and  there  is  evidence  of  its  presence  in  the  United 
States. 

Morphology. — The  micrococci  in  the  tissues  are  comparatively 
large — 1  to  1.5  ^  in  diameter.  They  are  embedded  in  the  thick 
capsules  of  the  organism,  forming  a  gelatinous  mass  of  consider- 
able size — a  zooglea.  Upon  culture-media  the  capsule  formation 


SPECIFIC   INFECTIOUS   DISEASES   PRODUCED   BY   COCCI      227 

is  not  very  evident,  if  present,  and  the  cells  are  usually  in  pairs  or 
tetrads.  There  is  some  evidence  that  this  organism  should  be 
grouped  as  a  Sarcina,  rather  than  as  a  Micrococcus,  while  in  many 
respects  it  resembles  the  Micrococcus  aureus.  It  stains  readily 
with  anilin  dyes,  and  is  gram-positive. 

Isolation  and  Culture. — M.  ascoformans  may  be  isolated  in 
pure  culture  from  the  characteristic  lesions,  or  in  mixed  infections 
it  may  be  secured  by  plating.  In  most  of  its  cultural  characters 
it  resembles  a  weakened  strain  of  M.  aureus.  Gelatin  colonies 
are  small,  and  cause  slight  liquefaction  and  cupping  of  the  medium 
when  at  the  surface.  In  gelatin  stab  the  growth  is  filiform  and 
white,  followed  by  a  slow,  crateriform  liquefaction.  The  colonies 
on  agar  are  scarcely  visible  in  most  strains,  although  more  vigorous 
types  have  been  described.  On  potato  the  growth  is  yellowish  and 
has  an  aromatic  odor. 

Physiology. — This  organism  produces  a  small  amount  of  gelat- 
inase,  as  evidenced  by  the  liquefaction  of  the  gelatin. 

Pathogenesis. — Experimental  Evidence. — Guinea-pigs  injected 
with  M.  ascoformans  generally  succumb  to  septicemia.  Mice 
are  not  susceptible.  Sheep  and  goats  develop  ulcers  at  the  point 
of  inoculation.  Injection  into  the  horse  usually  results  in  sup- 
puration, but  occasionally  the  typical  botryomycomata  are  de- 
veloped. Whether  or  not  this  is  a  variety  of  M.  aureus  is 
unsettled,  but  it  seems  probable  that  it  is  a  distinct  species. 

Character  of  Disease  and  Lesions  Produced. — The  lesions 
resemble  superficially  certain  fibromata  and  other  neoplasms. 
The  infection  usually  takes  place  where  the  surface  of  the  skin 
has  been  abraded,  as  by  harness,  through  wounds  at  castration, 
on  the  udder,  and  elsewhere  on  the  body.  The  tissues  involved 
become  grayish-red,  then  lardaceous,  and  eventually  form  a  mass 
resembling  a  fibroma.  These  sometimes  reach  considerable  size. 
Metastatic  infection  through  the  lymph-channels  may  result  in 
the  involvement  of  considerable  areas  and  infection  of  the  internal 
organs,  particularly  the  lungs. 

Immunity. — Methods  of  developing  immunity  have  not  been  de- 
vised. It  is  possible  that  autogenic  vaccination  might  be  of  value. 

Transmission. — As  noted  above,  infection  generally  occurs 
through  wounds  or  abrasions  of  the  skin. 


CHAPTER  XXIII 

NON-SPECIFIC  PYOGENIC  BACILLI 

MANY  bacilli  have  been  isolated  from  suppurative  infections. 
Some  of  these  undoubtedly  have  no  causal  relationship  to  the  pus- 
production  and  are  purely  saprophytic;  others,  as  the  colon  bacil- 
lus, are  normally  non-pathogenic,  but  under  certain  conditions 
may  cause  pus-infection;  others,  as  the  typhoid  bacillus,  normally 
cause  specific  diseases,  but  occasionally  produce  a  secondary 
pyogenic  infection,  while  others  are  known  only  from  their  associa- 
tion with  suppurative  processes,  and  may  be  termed  true  pyogenic 
bacilli.  Two  organisms  belonging  to  the  latter  class  are  worthy 
of  specific  mention — Bacillus  pyocyaneus  and  B.  pyogenes  suis. 
They  are  grouped  here  solely  because  of  their  pyogenic  properties, 
and  not  on  account  of  any  relationships  existing  between  them. 
Their  only  common  characteristics  are  their  shape  and  ability  to 
incite  suppuration.  Our  knowledge  of  the  B.  pyogenes  suis  is 
unsatisfactory.  It  probably  should  be  grouped  with  some  other 
forms,  possibly  with  the  Bacillus  pseudotuberculosis. 

Bacillus  pyocyaneus 

Synonyms. — Pseudomonas  pyocyanea;  Ps.  aeruginosa;  bacillus 
of  green  or  blue-green  pus  in  man  and  animals. 

Gessard,  in  1882,  described  the  Bacillus  pyocyaneus  from  blue- 
green  pus.  Since  that  time  it  has  been  isolated  and  studied  by 
numerous  investigators,  both  in  Europe  and  America. 

Distribution. — This  organism  has  been  isolated  from  the  feces 
of  man  and  animals,  from  sewage  and  surface  waters,  from  the 
soil,  and  from  air  and  dust.  It  is  usually  saprophytic  or  com- 
mensal in  its  growth,  and  is  only  rarely  pathogenic. 

Morphology  and  Staining. — B.  pyocyaneus  is  a  slender  rod  with 
rounded  ends,  about  0.6  by  2.6  u  or  smaller,  usually  single,  rarely 
in  chains  of  2  to  6  individuals.  It  is  motile  by  means  of  a  single 
terminal  flagellum.  No  spores  or  capsules  are  produced.  It 
stains  readily  by  the  ordinary  anilin  dyes,  and  is  gram-negative. 

228 


NON-SPECIFIC   PYOGENIC   BACILLI  229 

Isolation  and  Culture. — This  organism  is  readily  isolated  by 
plating  out  pus  which  contains  it,  as  the  colonies  are  quite  charac- 
teristic on  account  of  the  green  pigment  which  they  diffuse. 
It  grows  readily  on  all  the  common  laboratory  media.  Upon 
agar  and  gelatin  plates  the  thin,  poorly  denned  colonies  are  charac- 
terized by  the  fluorescent  pigment  surrounding  them.  Upon 
slant  agar  the  color  diffuses  until  the  whole  of  the  medium  is  a 
light  green,  then  a  darker  blue-green,  and  finally  a  brown  or  brown- 
red.  Gelatin  is  rapidly  liquefied.  Bouillon  is  clouded,  a  pellicle 
forms,  and  the  fluorescent  pigment  diffuses  from  the  top  downward. 
Potatoes  support  a  slimy  growth 
and  turn  green,  then  brown. 
Milk  is  coagulated  by  a  rennet- 
like  enzyme,  and  the  curd  pep- 
tonized. 

Physiology. — This  organism 
is  preferably  an  aerobe,  and 
grows  most  luxuriantly  in  the 
presence  of  oxygen,  but  growth 
will  continue  under  anaerobic 
conditions.  Proteolytic  fer- 
ments which  will  digest  gelatin, 

rig.  97. — Bacillus  pyocyaneus  (Kolle 
fibrin,  and  casein  are  produced.  and  Wassermann). 

Two      pigments     are     usually 

formed — one  green  and  fluorescent  (fluorescin),  the  other  (pyo- 
cyanin)  bluish.  Pigment  is  not  produced  in  the  absence  of 
oxygen.  In  old  cultures  the  pigments  become  yellow  or  brown. 
Autolytic  disintegration  of  the  cells  takes  place  in  old  cultures. 
Mucin,  a  compound  made  up  of  a  protein  and  a  carbohydrate, 
has  been  found  present  in  cultures.  To  this  may  be  ascribed  its 
slimy  consistency  on  agar  or  even  in  bouillon.  The  organism  is 
resistant  to  desiccation. 

Pathogenesis. — Experimental  Evidence. — Injection  of  cultures 
of  B.  pyocyaneus  subcutaneously  into  the  guinea-pig  or  rabbit 
causes  rapidly  spreading  edema,  suppuration,  septicemia,  and  death 
within  a  day  or  two.  Not  all  cultures  are  equally  pathogenic. 

Character  of  Infection  Produced. — The  B.  pyocyaneus  is  usually 
a  secondary  invader,  although  in  man  it  has  been  found  causing 


230  VETERINARY   BACTERIOLOGY 

primary  infections.  It  has  not  yet  been  proved  ever  to  cause 
suppuration  alone  in  any  of  the  domestic  animals,  but  is  not  un- 
common in  pus,  to  which  it  gives  a  green  or  blue-green  color. 
In  man  it  has  been  found  in  purulent  otitis  media,  meningitis, 
bronchopneumonia,  infantile  diarrhea,  and  generalized  infections. 

Immunity. — A  true  toxin  is  produced  by  virulent  cultures. 
Wassermann  found  0.2  to  0.5  c.c.  of  this  fatal  for  the  guinea-pig. 
An  antitoxin  has  been  prepared  for  this  pyocyaneus  toxin.  An 
endotoxin  has  also  been  demonstrated.  A  leukocytic  poison,  leU- 
kocidin,  and  a  hemolytic  toxin,  hemotoxin,  have  been  differentiated. 
Immunity  has  been  experimentally  produced  by  the  injection  of 
killed  cultures. 

Bacteriologic  Diagnosis. — The  organisms  may  be  most  readily 
determined  by  plating.  The  presence  of  a  gram-negative  bacillus 
in  pus  in  a  stained  mount  constitutes  presumptive  evidence. 

Bacillus  pyogencs  sois 

This  organism  has  been  several  times  described  from  inflam- 
matory suppurative  processes  in  the  hog. 

Morphology  and  Staining. — The  organism  is  a  slender,  non- 
motile  bacillus.  It  produces  neither  spores  nor  capsules.  It 
stains  readily  with  the  ordinary  anilin  dyes.  It  is  gram-negative. 

Isolation  and  Culture. — The  organism  may  be  readily  isolated 
in  pure  cultures  immediately  from  the  pus.  It  grows  readily 
on  artificial  media,  particularly  on  coagulated  blood-serum. 
On  this  latter  medium  the  small  dry  colonies  form  a  slight  area  of 
liquefaction  immediately  about  them. 

Physiology. — Growth  occurs  best  at  37°.  No  gas  is  produced 
from  dextrose. 

Pathogenesis. — The  organism  has  been  found  associated  com- 
monly with  suppurative  processes  in  the  hog,  particularly  those 
affecting  the  serous  membranes  lining  the  body-cavities.  These 
abscesses  are  generally  encapsulated.  'Evidently  they  may  arise 
as  metastatic  infections,  for  they  are  sometimes  distributed 
throughout  the  body.  The  organism  is  pathogenic  to  rabbits 
and  mice,  and  may  set  up  inflammatory  processes  in  these  ani- 
mals closely  resembling  those  of  the  hog. 


CHAPTER  XXIV 

DIPHTHERIA  GROUP 

Two  organisms  will  be  considered  under  this  heading,  the 
Bacillus  diphtheria  and  B.  pseudodiphthericus.  The  former  is  of 
importance  in  human  medicine  as  the  cause  of  the  disease  diph- 
theria, and  to  the  veterinarian,  because  of  the  use  of  horses  and 
other  animals  in  the  manufacture  of  antitoxin,  and  because  of  the 
use  that  has  been  made  of  the  toxin  in  the  development  of  the 
theories  of  immunity.  The  B.  pseudodiphihericus  is  important 
because  of  its  resemblance  to  the  diphtheria  bacillus. 

The  characters  which  particularly  differentiate  this  group  are 
the  polar  or  banded  staining  of  the  organisms  (presence  of  meta- 
chromatic  granules)  and  the  production  of  the  characteristic 
toxin  by  the  diphtheria  bacillus. 

The  term  diphtheria  is  used  in  a  pathologic  sense  to  denote  a 
type  of  inflammation  characterized  by  more  or  less  extensive 
necrosis,  and  the  formation  of  false  membranes  of  fibrin,  which  are 
rather  intimately  joined  to  the  tissue  which  produces  them. 
Many  organisms  can  bring  about  diphtheritic  inflammation  in  the 
tissues  of  man  and  animals.  These  organisms  belong  to  such 
varied  genera  as  Streptococcus,  Bacillus,  and  Actinomyces;  a 
pathologic  grouping  w^ould,  therefore,  bring  together  unrelated 
forms,  hence  the  inclusion  of  but  the  one  organism  under  this 
group.  By  diphtheria  is  meant  the  specific  disease  of  man  caused 
by  the  Bacillus  diphtheria,  and  diseases  of  animals  resembling  it 
clinically,  as  fowl  diphtheria,  are  not  to  be  confused  with  it. 

Bacillus  diphtherias 

Synonyms. — Klebs-Loffler  bacillus;  Bacterium  diphtheria;  Cor- 
ynebacterium  diphtheria;  Mycobacterium  diphtheria. 

Disease  Produced. — Diphtheria  in  man,  rarely  in  some  animals. 
Klebs,  in  1883,  described  an  organism  present  in  the  false  mem- 

231 


232  VETERINARY   BACTERIOLOGY 

brane  of  diphtheria,  which  Loffler,  in  1884,  secured  in  pure  cul- 
ture and  showed  to  be  pathogenic.  A  similar  organism  was 
isolated  by  him  from  a  healthy  child,  so  that  he  was  reluctant  to 
conclude  that  he  had  found  the  true  cause  of  the  disease.  Roux 
and  Yersin,  in  1888-1890,  showed  that  the  various  pathologic 
conditions  most  characteristic  of  diphtheria  could  be  duplicated 
in  animals  by  injection  of  the  broth  filtrate  containing  the  toxin. 

Distribution. — Occurs  in  epidemics,  particularly  among  the 
young,  in  Europe  and  America. 

Morphology  and  Staining. — Bacillus  diphtheric?  is  so  variable 
in  its  morphology  that  many  writers  do  not  consider  it  a  Bacillus 


,. 

+  .  ^ 


Fig.  98. — Bacillus  diphtheria  (Epstein  in  Journal  of  Infectious  Diseases). 

at  all,  but  to  be  more  closely  related  to  some  of  the  higher  bacteria 
or  even  the  fungi.  It  stains  readily  with  the  .common  anilin  dyes, 
and  is  gram-positive.  When  stained  with  methylene-blue  a  smear, 
prepared  directly  from  an  infected  mucous  membrane,  will  show 
rods  varying  from  0.4  to  1  /tf  in  diameter  and  1.5  to  3.5  fi  in  length, 
frequently  slightly  curved,  sometimes  pointed  or  club-shaped, 
sometimes  staining  uniformly,  but  usually  containing  meta- 
chromatic  granules,  which  stain  more  deeply  than  the  remainder 
of  the  cell,  and  give  a  barred  or  granular  appearance  to  the  cell 
contents.  These  same  variations  may  be  observed  in  the  organism 
taken  from  suitable  culture-media,  particularly  Loffler's  blood- 


DIPHTHERIA   GROUP  233 

serum.  Occasionally  branched  forms  may  be  observed.  Demmy 
has  shown  that  the  Bacillus  diphtherias  varies  almost  from  hour 
to  hour  in  its  morphology  when  grown  upon  blood-serum.  In 
five  hours  after  the  culture  is  made  the  cells  take  the  stain  uni- 
formly; in  eight  hours  some  cells  show  vacuolization;  in  twelve 
hours  the  organism  is  larger  and  stains  unevenly,  and  within 
forty-eight  hours  irregular  and  clubbed  forms  are  abundant. 
Wesbrook  has  constructed  a  chart  which  is  in  common  use  in 
laboratories  in  the  designation  of  the  different  types  of  bacilli 
to  be  observed.  Upon  media  where  growth  occurs  more  slowly 
than  upon  serum  the  organism  remains  smaller  and  stains  more 


Fig.  99. — Bacillus  diphtheria,  Wesbrook's  types:  a,  c,  d,  Granular  types;  a1,  c1, 
d1,  barred  types;  a2,  c2,  d2,  solid  types  (X  1500)  (McFarland). 

uniformly.  Whether  or  not  the  morphologic  types  of  Wesbrook 
represent  true  varieties  or  differ  in  their  pathogenic  properties 
is  a  matter  of  dispute.  Wesbrook  claims  the  types  which  develop 
rapidly  upon  blood-serum  and  show  distinct  granulation  are  viru- 
lent, while  the  slower  growing  solid  types  are  relatively  non- 
virulent.  It  is  claimed  by  others  that  the  latter  simply  represent 
the  B.  pseudodiphthericus,  the  organism  next  to  be  considered. 
No  spores  or  capsules  are  produced.  The  organism  is  non-motile. 
Isolation  and  Culture. — Bacillus  diphtheria  may  usually  be 
isolated  directly  from  the  throat  of  a  diphtheritic  patient  in  pure 
culture  upon  agar  or,  better,  upon  blood-serum.  In  mixed  infec- 


234 


VETERINARY   BACTERIOLOGY 


tions  glycerin-agar  plates  may  be  poured  from  the  growth  on 
blood-serum.  It  grows  well  upon  most  of  the  laboratory  media. 
Upon  Loffler's  blood-serum  distinct  colonies  are  sometimes  visible 
within  twelve  hours  as  minute,  pin-point,  translucent  dots;  these 
enlarge,  and  within  twenty-four  hours  show  as  small,  opaque, 
gray  colonies,  usually  discrete.  The  organism  develops  somewhat 
less  luxuriantly  upon  agar  and  gelatin,  although  repeated  trans- 
fers tend  to  increase  the  luxuriance.  Growth  occurs  in  milk, 
with  but  little  or  no  observable  change  in  the  medium.  Broth 
may  be  clouded.  A  delicate  film  or  pellicle  forms  on  the  surface 
after  a  time,  and  if  transfers  of  this  are  made  to  fresh  broth,  the 

growth  may  be  largely  con- 
fined to  pellicle  production. 
Advantage  is  taken  of  this 
fact  in  growing  the  organ- 
isms in  production  of  diph- 
theria toxin. 

Physiology. — This  or- 
ganism is  aerobic.  Upon 
culture-media  it  will  remain 
alive  for  long  periods. 
Desiccation  of  a  diphther- 
itic membrane  does  not 
necessarily  destroy  the 
bacillus,  and  it  has  been 
isolated  from  such  after  a 
period  of  months.  The 
heat  of  pasteurization,  60° 

for  thirty  minutes,  destroys  it.  In  the  dried  condition  it  is  more 
resistant.  Dextrose  is  fermented  by  virulent  forms  in  general, 
with  the  production  of  acid,  but  no  gas.  Lactose  is  not  fer- 
mented. The  non-virulent  types  are  believed  to  show  less  power 
of  acid  production.  Nitrates  are  reduced  to  nitrites.  No  indol 
is  produced.  Proteolytic  enzymes  are  not  formed;  gelatin  is  not 
liquefied. 

Pathogenesis. — Mechanism  of  Disease  Production. — Diphtheria 
is  one  of  the  best  examples  of  a  toxemia,  as  the  organism  remains 
upon  the  surface  of  the  mucous  membranes  and  in  the  necrotic 


Fig.  100. — Bacillus  diphtherias,  mount 
from  blood-serum  showing  the  character- 
istic metachromatic  granules  (Frankel  and 
Pfeiffer). 


DIPHTHERIA    GROUP 


235 


tissue,  rarely,  if  ever,  entering  the  blood-stream,  and  brings  about 
the  characteristic  lesions  of  the  disease  by  means  of  the  toxins 
which  it  produces.  These  are  absorbed  into  the  blood  and  bring 
about  changes  in  many  of  the  organs  of  the  body.  Death  is 
sometimes  due  directly  to  asphyxiation  by  the  false  membranes 
occluding  the  air-passages. 

Experimental  Evidence  of  Pathogenesis. — The  typical  diphtheritic 
inflammation  of  the  mucous  membranes  may  be  reproduced  in 
young  animals  by  direct  inoculation  in  the  trachea,  and  in  older 
animals  as  well  if  the  membranes  are -injured  previously.  The 
histologic  lesions  of  the  body  organs  can  be  produced  by  injections 
of  the  toxin  of  the  organ- 
ism. Paralysis  due  to  tox- 
one  poisoning  may  be 
developed  under  certain 
conditions.  That  this  or- 
ganism is  the  specific  cause 
of  diphtheria  is,  therefore, 
definitely  established. 

Character  of  Disease  and 
Lesions  Produced.  —  The 
pharynx  is  most  commonly 
affected;  the  larynx  and 
the  nasal  mucous  mem- 
branes are  sometimes  in- 
volved. There  may  also 
be  diphtheritic  conjunctiv- 
itis, diphtheritic  infections  of  the  middle  ear  and  of  the  mucous 
membranes  of  the  genital  organs.  The  organisms  remain  localized, 
rarely  entering  the  blood-stream,  cause  necrosis  and  degenera- 
tion of  the  epithelial  cells  and  the  deeper  tissues  as  well.  Blood- 
serum  and  fibrin  are  exuded  from  the  vessels,  and,  together 
with  fragments  of  the  necrosed  tissue,  form  the  diphtheritic 
membrane.  Acute  interstitial  nephritis,  fatty  degeneration  of  the 
myocardium,  and  sometimes  of  the  nerve  tissues,  are  due  to  the 
absorption  of  the  toxins.  There  is  no  constant  ratio  between 
the  extent  of  the  diphtheritic  process  in  the  throat  or  elsewhere  and 
the  severity  of  the  symptoms  and  lesions  produced  by  the  toxin. 


Fig.  101. — Bacillus  diphtheria,  twenty- 
four-hour  colony  on  agar  ( X  100)  (Frankel 
and  Pfeiffer). 


236  VETERINARY   BACTERIOLOGY 

Immunity. — The  diphtheria  bacillus,  according  to  Ehrlich, 
produces  two  types  of  poison,  the  true  toxin  and  the  toxone.  The 
first  is  responsible  for  the  acute  symptoms  of  poisoning,  the  latter 
for  the  paralysis  that  frequently  occurs  during  convalescence. 
The  haptophores  of  both  toxin  and  toxone  are  believed  to  be 
identical,  and  to  be  neutralized  by  the  same  antitoxin.  A  con- 
sideration of  the  production  of  diphtheria  antitoxin  has  been  taken 
up  in  the  chapter  on  Toxins  and  Antitoxins,  under  the  heading  of 
Immunity.  Agglutinins  may  develop  in  the  blood,  but  are  not 
constant,  and  are  of  no  practical  value  in  diagnosis.  They  may 
be  produced  in  experimental  animals  by  the  injection  of  killed 
cultures  of  the  diphtheria  bacillus.  Precipitins  have  likewise 
been  produced  by  artificial  immunization,  but  usually  cannot  be 
demonstrated  in  the  blood  of  infected  individuals.  Bactericidal 
sera  may  be  prepared  by  repeated  injections  of  killed,  washed 
cultures  of  diphtheria  bacilli,  and  favorable  results  have  been 
reported  in  the  use  of  such  a  serum  in  freeing  the  throat  of  con- 
valescents from  the  bacteria.  Its  clinical  value  can  scarcely  be 
said  to  be  proved.  Opsonins  have  not  been  demonstrated. 

The  value  of  diphtheria  antitoxin  for  prophylactic  and  curative 
injections  is  well  established.  It  has  resulted  in  materially  lessen- 
ing mortality  whenever  it  has  been  used.  As  much  as  100,000 
units  have  been  injected  in  some  cases,  but  the  usual  curative 
dose  is  8000  to  15,000  units. 

Bacteriologic  Diagnosis. — Sterile  swabs  are  used  to  swab  out  the 
throat  and  nose  of  the  suspect,  and  are  smeared  over  the  surface 
of  blood-serum.  Mounts  made  in  five  to  eighteen  hours  thereafter, 
stained  with  Lofner's  methylene-blue,  should  show  the  character- 
istic organism  with  its  metachromatic  granules.  Diagnosis  may 
sometimes  be  made  directly  from  smears  taken  from  the  throat. 

Transmission. — The  organism  doubtless  sometimes  gains 
entrance  into  the  body  by  the  inhalation  of  infective  droplets,  but 
more  commonly  by  the  use  of  common  drinking  vessels  and 
through  fomites. 

Bacillus  pseudodiphthericus 

Synonyms. — Bacterium  pseudodiphthericum ;  Mycobaderium 
pseudodiphthericum;  bacillus  of  Hoffman;  Bacillus  clavatus; 
Corynebacterium  pseudodiphthericum. 


DIPHTHERIA   GROUP  237 

Loffler,  in  1887,  found  an  organism  in  diphtheritic  membranes 
that  resembled  the  true  diphtheria  bacillus  closely.  Hoffman,  a 
little  later,  made  a  series  of  similar  observations.  These  organisms 
have  since  been  found  repeatedly  in  normal  throats,  as  well  as 
associated  with  the  true  diphtheria  bacillus  in  disease. 

Distribution. — It  has  been  estimated  that  about  one-sixth  of  all 
healthy  individuals  have  the  pseudodiphtheria  bacilli  present  in 
the  mouth  and  throat. 

Morphology. — Morphologically,  some  races  have  been  dis- 
covered that  are  practically  indistinguishable  from  the  B.  diph- 
theria. In  general,  however,  the  pseudodiphtheria  organism  is 
somewhat  shorter  and  plumper,  and  does  not  ordinarily  show 
granules  when  treated  with  the  Neisser  stain.  Some  investiga- 
tors claim  to  have  observed  transformations  of  the  one  type  into 
the  other,  but  these  statements  need  verification,  for  other  careful 
workers  have  failed. 

Pathogenesis. — The  principal  differential  character  is  the  lack 
of  pathogenesis  and  toxin-production  by  the  Hoffman  type. 
Transformation  of  the  non-virulent  into  the  virulent  type  has  not 
been  satisfactorily  demonstrated.  The  organism  is  of  importance 
only  in  that  it  may  lead  to  a  mistaken  diagnosis  of  diphtheria. 


CHAPTER  XXV 

BACILLUS  PSEUDOTUBERCULOSIS  GROUP 

THE  term  "  pseudotuberculosis  bacillus  "  is  applied  to  any 
organism  that  produces  a  disease  in  which  nodules  resembling 
those  of  tuberculosis  are  formed.  The  name  does  not  refer  to  any 
relationship  of  the  organism  to  the  Bacillus  tuberculosis,  but 
simply  to  the  similarity  of  the  lesions  produced.  These  organisms 
are  not  acid  fast.  They  all  resemble  each  other  in  being  the  cause 
of  chronic  caseations  and  suppurations,  particularly  of  the  lymph- 
nodes. 

This  group  of  organisms,  including  the  B.  pseudotuberculosis, 
B.  lymphangitidis  ulcer osa,  and  B.  pyogenes  bovis,  is  in  need  of 
careful  revision,  for  the  species  limits  are  not  well  understood. 
These  organisms  all  resemble  the  diphtheria  bacillus  somewhat, 
particularly  in  pleomorphism,  shape,  and  staining  characters. 
None,  however,  are  known  to  produce  any  toxins. 

Bacillus  pseudotuberculosis 

Synonyms. — Bacillus  pseudotuberculosis  ovis;  Mycobacterium 
pseudotuberculosis;  Bacillus  tuberculosis  murium;  bacillus  of 
Preisz. 

Diseases  Produced. — Caseous  lymphadenitis  in  sheep,  and 
similar  infections  in  the  mouse  and  rarely  in  cattle. 

Organisms  differing  morphologically  from  the  tubercle  bacillus, 
but  causing  somewhat  similar  lesions  in  the  body,  were  first  noted 
by  Malassez  and  Vignal  in  1883.  Charrin  and  Roger,  in  1888, 
described  nodules  in  the  liver  and  spleen  of  guinea-pigs,  caused  by 
organisms  that  were  not  acid  fast.  Nocard,  in  1889,  studied  an 
epizootic  among  rabbits  caused  by  a  similar  organism.  Preisz 
and  Guinard,  in  1891,  reported  pseudotuberculosis  in  sheep,  and 
in  1894  Preisz  published  an  extended  account  of  the  disease. 
In  the  United  States  Norgaard,  in  1899,  published  an  account  of 


BACILLUS   PSEUDOTUBERCULOSIS   GROUP 


239 


the  disease  and  its  organism,  which  showed  it  to  be  of  considerable 
economic  importance  in  sheep  and  to  be  quite  generally  distributed. 
This  organism  is  not  to  be  confused  with  the  pseudotuberculosis 
bacillus  of  Pfeiffer  infecting  guinea-pigs.  The  latter  is  wholly 
different,  and  belongs  to  another  group  entirely. 

Distribution. — The  disease  in  sheep  has  been  reported  from 
Europe,  South  America,  and  North  America,  particularly  in  aged 
sheep. 

Morphology. — Bacillus  pseudotuberculosis  is  a  short,  straight 
rod,  with  rounded  ends,  quite  variable  in  size,  about  0.4  ^  by 
1.3  to  1.6  u  or  longer.  Sometimes  clubbed  types  are  observed,  the 
enlarged  portion  staining  more  intensely  than  the  remainder, 


Fig.    102. — Bacillus  pseudotubercvlosis,   colony  and  mount    (Norgaard   and 
Mohler  in   Report  of   Bureau  of  Animal  Industry). 

reminding  one  of  somewhat  similar  types  in  the  B.  diphtheria. 
Chain  formation  rarely  occurs;  the  organisms  are  usually  found 
in  pairs.  It  is  non-motile,  and  produces  no  spores  or  capsules. 
It  stains  readily  with  the  ordinary  anilin  dyes,  and  is  gram- 
positive. 

Isolation  and  Culture. — Cultures  may  be  obtained  in  pure 
condition  by  opening  a  caseous  nodule  and  making  smears  upon 
agar.  Growth  is  scant  at  first,  but  becomes  better  after  a  time,  as 
the  organism  adapts  itself  to  growth  on  artificial  media.  The 
discrete  colonies  produced  by  the  initial  inoculation  grow  in  twelve 
days  to  a  diameter  of  4  to  6  mm.;  they  are  rounded,  thick,  gray- 
white  in  color,  with  a  waxy  and  granular  surface,  and  with  con- 


240  VETERINARY   BACTERIOLOGY 

centric  rings  and  a  papillate  center.  In  subsequent  transfers 
the  colonies  are  confluent,  dry,  and  cohesive.  Glycerin  agar 
is  not  so  favorable  as  the  plain  agar.  Bouillon  becomes  turbid, 
then  clears  by  sedimentation,  and  a  white,  "  greasy,"  brittle 
pellicle  forms.  Gelatin  is  not  a  suitable  medium,  as  little  growth 
occurs  at  room-temperatures.  Blood-serum  is  favorable,  the 
colonies  frequently  becoming  creamy  yellow  in  color.  Growth 
occurs  on  potato  and  in  milk.  In  the  latter  the  appearance  is 
unchanged.  The  organism  described  by  Preisz  is  recorded  as 
showing  some  minor  differences  in  cultural  characters  from  the 
one  studied  by  Norgaard. 

Physiology. — The  organism  is  aerobic  and  facultative  anae- 
robic. A  little  acid  is  formed  from  dextrose,  .but  none  from 
lactose  or  saccharose.  No  gas  is  developed.  Neither  indol  nor 
phenol  is  produced.  The  growth  optimum  is  37°,  although  some 
growth  will  occur  at  16°  to  18°.  The  organism  will  resist  desicca- 
tion for  several  days.  It  is  easily  destroyed  by  disinfectants. 

Pathogenesis. — Mechanism  of  Disease  Production. — The  disease 
may  be  characterized  as  a  chronic  caseation  of  the  lymph-nodes, 
without  general  systemic  disturbances.  No  toxins  are  produced, 
and  the  means  by  which  the  organism  brings  about  its  local 
reaction  is  unknown. 

Experimental  Evidence  of  Pathogenesis. — Intravenous  injec- 
tion of  the  guinea-pig  results  in  death  in  four  to  ten  days,  with 
foci  of  caseation  in  various  internal  organs,  particularly  the  lungs 
and  the  liver.  Subcutaneous  injection  is  followed  by  enlarge- 
ment and  caseation  or  suppuration  of  the  lymph-glands,  result- 
ing fatally  in  from  fifteen  to  twenty-eight  days.  Feeding  experi- 
ments have  been  successful  in  causing  infection.  Similar  results 
may  be  reached  by  the  use  of  the  rabbit,  the  mouse,  but  not 
pigeons  and  fowls.  Experimental  inoculation  of  sheep  has  repro- 
duced the  disease. 

Character  of  Disease  and  Lesions  Produced. — The  organism  has 
been  reported  chiefly  as  a  cause  of  ovine  caseous  lymphadenitis, 
but  an  organism  probably  identical  with  this  form  has  been 
reported  from  similar  lesions  in  the  lungs  of  cattle.  It  is  possible 
that  it  likewise  is  identical  with  the  type  next  to  be  described. 
The  disease  of  sheep  is  found  chiefly  in  breeding  ewes.  The 


BACILLUS   PSEUDOTUBERCULOSIS   GROUP  241 

disease  progresses  slowly,  and  is  frequently  not  recognized  until 
the  animal  is  slaughtered.  The  lymphatic  glands  are  most  fre- 
quently affected.  These  glands  enlarge,  caseate,  and  are  often 
encapsulated.  In  more  advanced  cases  the  internal  organs  are 
also  infected,  nodules  appearing  in  the  lungs,  spleen,  and  liver, 
and  sometimes  in  the  kidneys.  The  lesions  are  not  commonly 
found  in  young  animals,  probably  because  the  disease  has  not 
had  time  to  develop  sufficiently  to  cause  the  enlarged  glands  to 
be  noted  in  inspection. 

Immunity. — The  organism  does  not  produce  a  toxin.  Ag- 
glutinin,  bacteriolysin,  and  opsonin  production,  as  well  as  methods 
of  immunization,  have  not  been  investigated. 

Bacteriologic  Diagnosis. — Mounts  from  pus  or  the  caseated  con- 
tents of  the  nodules,  stained  by  Gram's  method,  should  show  the 
characteristic  bacillus.  The  use  of  the  acid-fast  staining  method 
will  differentiate  from  B.  tuberculosis.  Isolation  upon  agar  slants 
may  sometimes  be  necessary  to  complete  the  identification. 

Transmission. — The  normal  infection  atrium  in  the  sheep  is 
not  certainly  known.  Probably  the  organism  may  gain  entrance 
through  skin  abrasions,  and  possibly  by  inhalation  and  ingestion. 

Bacillus  lymphangitidis  ulcerosa 

Synonym. — Bacillus  of  pseudofarcy. 

Disease  Produced. — Ulcerative  lymphangitis,  pseudofarcy,  or 
pseudoglanders  in  equines. 

Nocard,  in  1892,  described  an  organism  which  he  believed 
to  be  the  etiologic  factor  in  an  outbreak  of  pseudofarcy  in  horses. 
Later  (1897)  he  concluded  that  his  organism  was  identical  with  the 
Bacillus  pseudotuberculosis  just  described.  Sufficient  differences 
in  the  organism  have  been  pointed  out  to  justify  the  retention  of  a 
separate  name  for  this  organism  until  further  investigations 
have  been  made.  The  probabilities  are  much  in  favor  of  the  iden- 
tity of  the  two  forms,  and  the  name  B.  pseudotuberculosis  might 
then  be  used  to  include  both. 

Morphology  and  Staining. — This  organism  resembles  the  pre- 
ceding morphologically.  It  is  gram-positive. 

Isolation  and  Culture. — The  organism  may  be  obtained  in  pure 
culture  directly  from  infected  lymph-glands.  Upon  agar  discrete, 

16 


242  VETERINARY   BACTERIOLOGY 

white,  opaque  colonies  are  formed.  These  generally  become  con- 
fluent. The  whole  mass  of  the  growth  may  be  detached  from  the 
surface  of  the  medium.  In  bouillon,  clouding  occurs,  then  the 
medium  clears  by  sedimentation,  with  or  without  a  pellicle.  Upon 
serum  the  organism  produces  outgrowths  into  the  medium,  and 
resembles  the  B.  pseudotuberculosis  closely.  Milk  is  not  changed. 

Physiology. — The  organism  is  aerobic.  Neither  acid  nor  gas 
is  developed  from  carbohydrates.  The  thermal  death-point  is 
about  65°  with  an  exposure  of  fifteen  minutes. 

Pathogenesis. — Experimental  Evidence. — Subcutaneous  inocu- 
lations of  the  guinea-pig  result  in  abscess  formation  and  extension 
along  the  lymph-channels.  Introduced  intraperitoneally  into  a 
male  guinea-pig,  it  commonly  produces  an  orchitis  which  cannot 
be  readily  differentiated  from  that  produced  by  the  glanders 
bacillus.  Nocard  reproduced  the  disease  in  the  horse  by  the 
inoculation  of  pure  cultures. 

Character  of  Lesions  and  Disease  Produced. — The  subcutaneous 
lymph-nodes  are  chiefly  affected.  They  enlarge,  and  break 
through  to  the  surface,  producing  an  abscess  characterized  by 
suppuration.  The  clinical  ^picture  closely  resembles  that  of  farcy. 
Involvement  of  the  deeper  glands  and  of  the  internal  organs 
occurs  later  in  the  progress  of  infection. 

Bacteriologic  Diagnosis. — This  organism  may  be  differen- 
tiated from  the  true  glanders  bacillus  by  the  fact  that  it  is 
gram-positive,  while  the  latter  is  gram-negative.  The  pro- 
duction of  an  orchitis  in  the  male  guinea-pig  when  injected 
makes  it  necessary  that  mounts  stained  by  Gram's  method  be 
used  to  differentiate  from  B.  mallei.  In  doubtful  cases,  pure 
cultures  should  be  grown  upon  the  potato. 

Immunity. — Nothing  is  known  of  the  metabolic  products  of 
the  growth  of  the  organism  nor  of  methods  of  immunization  or 
of  the  body  reactions  to  its  presence. 

Transmission. — The  disease  is  probably  transmitted  through 
skin  lesions,  possibly  by  inhalation  or  ingestion. 

Bacillus  pyogcnes  bovis 

Synonyms. — Bacillus  renalis  bovis;  B.  pyelonephritidis  bovis. 
Infection    Produced. — Pyelonephritis,     caseation    of    lymph- 
glands,  and  chronic  pneumonia  in  cattle. 


BACILLUS   PSEUDOTUBERCULOSIS   GROUP  243 

It  is  probable  that  this  organism  is  identical  with  the  Bacillus 
pseudotuberculosis  discussed  above  or  at  least  closely  related  to 
it.  It  has  been  isolated  from  cattle  by  several  investigators. 

Distribution. — This  organism  has  been  reported  both  from  this 
country  and  Europe. 

Morphology  and  Staining. — This  organism  resembles  the  Bacil- 
lus pseudotuberculosis  in  many  respects,  but  is  somewhat  larger — 
0.7  by  2  to  3.8  /^.  It  is  sometimes  granular,  and  produces  clubbed 
or  even  branched  forms.  It  is  quite  commonly  bent.  It  is  non- 
motile,  does  not  produce  spores  or  capsules.  It  stains  readily 
with  the  common  anilin  dyes,  and  is  gram-positive. 

Isolation  and  Culture. — The  organism  may  be  isolated  in  pure 
culture  from  infected  lymph-nodes  or  other  lesions  directly  upon 
agar.  Agar  slants  show  discrete,  grayish-white  colonies  that 
never  become  large.  From  the  periphery  of  some  of  these  colonies 
short  filaments  radiate  into  the  agar.  Bouillon  remains  clear,  and 
a  distinct  sediment  forms. 

Physiology. — This  organism  tends  to  die  out  quickly  in  arti- 
ficial media.  It  is  aerobic.  No  acids  nor  gas  are  produced  from 
carbohydrates. 

Pathogenesis. — Our  knowledge  of  the  pathogenesis  of  this 
organism  is  not  in  a  satisfactory  state.  It  has  been  isolated  from 
chronic  bronchopneumonia  in  cattle  and  from  various  other 
secondary  and  metastatic  infections.  It  has  also  been  found  asso- 
ciated with  pyelonephritis.  This  latter  infection  is  marked  by 
enlargement  of  the  kidneys  and  a  purulent  inflammation  of  the 
lining  mucous  membranes  of  the  ureters  and  bladder.  The  in- 
flammation may  cause  necrosis  of  the  tissues.  When  injected 
into  guinea-pigs  or  mice,  suppuration  may  be  induced. 

Immunity. — Nothing  is  known  relative  to  the  metabolic  prod- 
ucts of  the  organism  or  of  methods  of  either  passive  or  active 
immunization. 

Bacteriologic  Diagnosis. — The  presence  of  the  organism  may 
be  demonstrated  by  the  use  of  Gram's  stain  and  by  pure  culture 
methods. 

Transmission. — The  infection  atria  are  probably  the  respiratory 
tract  in  lung  infection  and  the  genito-urinary  organs,  particularly 
in  the  female  following  parturition. 


CHAPTER  XXVI 

SWINE  ERYSIPELAS  GROUP 

Two  organisms  have  been  described  as  belonging  to  this 
group — the  Bacillus  rhusiopathice  and  B.  murisepticus.  As  will 
be  noted  later,  there  is  good  reason  to  believe  that  these  organ- 
isms are  identical.  The  group  may  be  characterized  as  made 
up  of  very  minute,  slender,  non-motile,  non-spore-producing, 
gram-positive  rods. 

Bacillus  rhusiopathiae 

Synonyms. — Bacillus  rhusiopathioe  suis;  Bacillus  erysipelatis 
suis. 

Disease  Produced. — Swine  erysipelas,  red  fever  of  swine, 
rouget,  Rotlauf. 

Loffler  in  1885  first  described  the  organism  present  in  swine 
erysipelas.  The  disease  had  previously  been  differentiated  from 
anthrax  by  Pasteur  and  Thuiller. 

Distribution. — The  disease  has  been  reported  from  most  of  the 
European  countries,  and  an  organism  resembling  the  Bacillus 
rhusiopathice  has  several  times  been  reported  from  the  United 
States,  although  there  has  been  no  satisfactory  demonstration 
of  the  presence  of  the  disease  in  this  country.  In  Europe  the 
disease  is  of  considerable  economic  importance. 

Morphology  and  Staining. — Bacillus  rhusiopathice  is  a  slender 
rod,  variously  given  as  0.2  to  0.4  (J-  by  1  to  2  a,  usually  straight, 
but  sometimes  somewhat  curved,  single  or  in  chains.  It  is  non- 
motile,  does  not  produce  spores  or  capsules.  It  stains  readily 
with  the  anilin  dyes  and  is  gram-positive. 

Isolation  and  Culture. — The  organism  may  be  secured  in  pure 
culture  by  plating  the  blood  or  pulp  from  some  of  the  internal 
organs,  the  spleen  in  particular.  The  colonies  in  the  gelatin 
plate  appear  on  the  second  or  third  day,  and  are  quite  character- 

244 


SWINE    ERYSIPELAS   GROUP 


245 


•*> 


istic.  Each  colony  is  found  to  be  surrounded  by  a  zone  of  much- 
branched  threads.  They  permeate  the  medium,  and  are  not  found 
upon  the  surface,  as  is  the  case  with  anthrax  and  other  forms  which 
produce  colonies  with  filamentous  edges. 

Gelatin  stab  cultures  develop  only  below  the  surface  of  the 
medium,  showing  the  micro-aerophilic  or  semi-anaerobic  growth 
characters  of  the  organism.  The  mature  stab  has  the  appearance 
of  a  test-tube  brush,  streaks  and  disks  of  growth  radiating  from  the 
center.  Streak  cultures  do  not  develop  well  upon  agar  or  blood- 
serum  except  by  growth  under  anaerobic  conditions,  preferably 
by  absorption  of  the  oxygen  by  the 
alkaline  pyrogallate  method.  Bouillon 
is  clouded  and  produces  a  grayish- 
white  sediment.  Ordinarily  no  growth 
occurs  upon  potatoes,  even  under 
anaerobic  conditions. 

Physiology. — The  B.  rhusiopathice 
grows  better  anaerobically  than  aerob- 
ically.  It  is  unusually  resistant  for 
a  non-spore-producing  form.  Desic- 
cation frequently  fails  to  destroy  the 
organism  in  several  weeks.  The 
thermal  death-point  is  recorded  by 
some  authors  as  low  as  52°  for  fifteen 
minutes,  by  others  as  high  as  70°. 
The  optimum  growth  temperature  is 
37°,  but  growth  occurs  well  at  room- 
temperatures.  Gelatin  is  not  com- 
pletely liquefied,  but  generally  softened. 

Pathogenesis. — Experimental  Evidence. — Mice  die  of  septicemia 
when  inoculated  with  pure  cultures.  Death  occurs  usually  within 
four  days,  frequently  within  forty-eight  hours.  Field-mice  are 
immune,  as  are  guinea-pigs,  cattle,  horses  and  other  equines, 
dogs,  cats,  chickens,  and  geese.  Rabbits  inoculated  subcuta- 
neously  develop  an  edema  and  redness  at  the  point  of  inocula- 
tion, and  this  erysipelas-like  lesion  spreads  to  other  parts  of 
the  body  and  the  animal  dies.  Intravenous  injection  is  quickly 
fatal  through  the  development  of  a  septicemia.  The  white  rat, 


Fig.  103. — Bacillus  rhusio- 
pathice, stab  culture  in  gelatin 
— four  days  (Frankel  and 
Pfeiffer). 


246  VETERINARY   BACTERIOLOGY 

the  sparrow,  and  the  pigeon  are  likewise  susceptible.  The  typi- 
cal disease  may  be  produced  in  swine  by  inoculation  of  pure  cul- 
tures. There  is  no  doubt  but  that  the  Bacillus  rhusiopathice  is 
the  cause  of  the  disease.  There  is  some  evidence  that  the  infec- 
tion in  a  mild  urticarial  form  is  occasionally  transmitted  to  the 
human. 

Character  of  Disease  and  Lesions  Produced. — Small  numbers  of 
bacteria  can  generally  be  demonstrated  in  the  blood,  and  abun- 
dantly in  various  of  the  body  organs,  particularly  the  spleen 
and  the  lungs.  In  the  acute  type  of  the  disease  the  mucous  mem- 
brane of  the  alimentary  tract  is  reddened,  and  shows  petechial 
hemorrhages.  The  spleen  and  the  lymph-nodes  are  somewhat 
enlarged,  and  the  lungs  are  usually  congested.  The  most  char- 
acteristic lesions  are  those  of  the  skin,  where  numerous  congested 
areas  and  local  hemorrhages  give  rise  to  the  spotted  appear- 
ance. In  chronic  cases  there  is  generally  a  verrucose  or  ulcerous 
endocarditis  which  is  quite  characteristic. 

Immunity. — No  true  toxin  has  been  demonstrated  for  the 
Bacillus  rhusiopathice.  The  presence  of  specific  amboceptors  in 
immune  serum  may  be  shown  by  the  method  of  complement 
absorption,  but  just  what  part  these  play  in  immunity  has  not 
been  satisfactorily  demonstrated.  Opsonins  are  present,  and  prob- 
ably account  in  part  for  the  immunity. 

Very  young  animals — under  three  months — and  those  over 
one  year  rarely  contract  the  disease.  Animals  that  recover  from 
the  disease  are  thereafter  immune. 

Active  Immunization. — It  has  been  shown  by  the  researches  of 
Pasteur,  later  by  Kitt  and  others,  that  passage  of  strains  through 
certain  animals,  particularly  the  rabbit  or  the  pigeon,  leads  to  an 
attenuation  such  that  the  material  may  be  used  for  vaccination 
of  the  hog.  It  is  also  known  that  continued  cultivation  usually 
reduces  the  virulence  of  the  organism  for  mice.  The  virulence 
is  subject  to  great  and  inexplicable  variations.  The  Pasteur 
vaccine  consists  of  an  attenuated  bouillon  culture  of  the  B.  rhusio- 
pathice. The  material  is  sent  out  in  two  packages,  labeled  Vaccine 
I.  and  Vaccine  II.  These  are  injected  fifteen  days  apart.  Im- 
munity is  established  in  two  to  three  weeks  after  the  second  injec- 
tion. The  injected  animals  sometimes  show  the  characteristic 


SWINE   ERYSIPELAS    GROUP  247 

urticaria  of  the  disease.  The  use  of  this  method  has  led  to  varied 
results  in  different  countries.  Voges  and  Schiitz  have  modified 
the  Pasteur  vaccine  method  by  doing  away  with  the  Vaccine  II., 
as  they  found  the  blood  still  contained  bacilli  at  the  time  of  the 
second  injection,  and  concluded  the  latter,  therefore,  useless. 
It  seems  that  a  satisfactory  immunization  of  the  hog  against  the 
disease  cannot  be  accomplished  by  injections  of  the  killed  organ- 
isms. 

Passive  Immunization. — Emmerlich  and  Mastbaum,  in  1891, 
described  a  method  of  preparing  an  immune  serum  which  would 
protect  animals  against  the  disease.  Their  method  was  impractical 
in  some  respects,  and  was  superseded  by  the  method  of  Lorenz,  and 
this  by  those  of  Leclainche,  Voges  and  Schiitz,  and  Lange.  The 
antiserum  is  prepared  by  the  intravenous  injection  of  virulent  bouil- 
lon cultures  into  the  horse.  Usually  100  c.c.  constitutes  the  initial 
injection,  and  is  followed  at  intervals  by  larger  amounts — up  to 
500  c.c.  The  injection  produces  a  rise  in  temperature,  and  other 
reactions  that  disappear  in  twenty-four  to  forty-eight  hours.  The 
immune  blood  is  drawn,  and  the  serum  used  for  passive  immuniza- 
tion of  swine.  It  is  prepared  on  a  large  scale  in  several  institutes 
in  Europe,  and  is  used  extensively.  Cattle  and  even  buffalo  are 
sometimes  used  instead  of  the  horse  in  the  preparation  of  the  serum. 
Prettner  claims  that  the  use  of  cattle  immune  serum  confers  a  more 
lasting  immunity  than  that  of  the  horse.  Schreuber  and  Schubert 
studied  the  question  of  multiplicity  of  amboceptors,  and  came  to 
the  conclusion  that  a  mixture  of  immune  sera  from  horses  and 
cattle  would  give  a  greater  variety  of  amboceptors  specific  for  the 
organism.  A  mixture  of  this  kind  is  termed  "  double  serum  " 
(German,  Rotlauf  Doppelserum).  The  double  serum  has  not 
proved  in  practice  to  be  of  any  greater  value  than  the  serum  from 
the  horse  alone.  The  immune  serum  is  usually  standardized  by 
the  use  of  the  mouse,  commonly  by  the  method  of  Lorenz.  A 
serum  suitable  for  use  should  immunize  a  mouse  in  doses  of  0.01 
gm.  per  10  gm.  of  weight,  against  injection  with  0.01  gm.  of  a 
virulent  culture.  Lorenz  used  mice  weighing  uniformly  15  gm. 
as  a  standard,  and  such  a  serum  is  said  to  have  a  titer  of  0.015  gm. 
(mouse).  Marx  has  modified  the  technic  of  Lorenz  somewhat, 
but  the  principle  is  the  same.  Leclainche  has  advised  the  use  of 


248  VETERINARY   BACTERIOLOGY 

the  pigeon,  rather  than  the  mouse,  as  a  test-animal.  The  serum 
may  be  used  curatively  or  prophylactically.  Amounts  up  to  30  c.c. 
are  used  in  curing  the  disease.  In  cases  not  too  far  advanced  it 
arrests  the  disease,  and  has  been  shown  to  reduce  the  mortality 
materially.  The  injection  of  the  serum  prophylactically  results 
in  temporary  immunity  only,  hence  it  is  customary  to  establish  an 
active  immunity  by  injection  of  the  specific  organism,  the  culture 
and  the  serum  being  mixed  together  or  injected  separately  at  the 
same  time.  This  method  of  immunization  has  proved  of  such 
value  that  it  is  extensively  used  in  Europe.  The  active  immunity 
developed  as  a  result  of  the  combined  method  lasts  for  periods  of 
six  months  to  a  year  or  even  longer. 

Bacteriologic  Diagnosis. — Smears  from  the  spleen,  sometimes 
from  the  blood,  will  show  the  characteristic  slender,  gram-positive 
bacillus.  Isolation  in  gelatin  plates  gives  a  characteristic  type  of 
colony.  The  stab  culture  in  gelatin  is  also  diagnostic. 

Transmission. — The  specific  organism  may  be  demonstrated 
in  the  feces  of  an  infected  individual.  It  may  gain  entrance  di- 
rectly through  the  skin,  but  probably,  in  most  instances,  the  infec- 
tion atrium  is  the  alimentary  tract.  Typical  virulent  bacilli  and 
non-virulent  forms  have  been  repeatedly  isolated  from  healthy 
animals.  It  is  evident  that  the  interrelationships  of  this  organism 
and  the  body  are  quite  complex. 

Bacillus    murisepticus 

Disease  Produced. — Mouse  septicemia. 

Koch,  in  1878,  called  attention  to  the  fact  that  the  injection 
of  putrid  meat  infusion  into  a  mouse  resulted  in  a  septicemia  due 
to  a  non-motile,  minute  rod.  It  is  of  importance  chiefly  because 
of  its  resemblance  to  the  Bacillus  rhusiopathice.  It  has  been 
isolated  from  a  variety  of  sources  in  nature,  and  has  been  known  to 
cause  epidemics  in  mice  kept  for  experimental  purposes.  There 
are  no  marked  cultural  characters  which  may  be  used  to  differen- 
tiate the  two  organisms,  and  morphologically  they  are  likewise 
very  similar.  It  is  claimed  by  some  that  the  B.  murisepticus  is 
somewhat  more  slender  than  the  B.  rhusiopathice.  It  has  been 
found  possible  to  immunize  the  rabbit  against  the  latter  by  injec- 
tions of  the  former.  However,  it  has  not  been  found  possible  to 


SWIXE    ERYSIPELAS    GROUP 


249 


produce  typical  swine  erysipelas  by  the  injection  of  B.  murisep- 
ticus.     It  is  undoubtedly  true  that  the  two  organisms  are  closely 


>•   - 


Fig.  104. — Bacillus  murisepticus,  stained  mount  (Giinther). 


Fig.  105. — Bacillus  murisepticus,  stab  culture  in  gelatin  (Giinther). 

related.      Whether  they  are  varieties  of  one  species,   differing 
in  virulence,  is  not  known. 


CHAPTER  XXVII 

GLANDERS  GROUP 

ONE  organism  only,  the  Bacillus  mallei,  the  cause  of  glanders 
and  farcy  in  equines,  is  known  to  belong  to  this  group.  It  should 
be  noted  that  the  so-called  pseudoglanders  and  the  causal  organ- 
isms are  treated  under  other  chapter  headings.  These  latter 
organisms  are  not  related  to  the  organism  in  question  except  in 
that  they  produce  lesions  which  are  sometimes  confused  with 
glanders  clinically.  Some  of  the  pseudoglanders  organisms  belong 
to  such  disease  groups  as  the  bacteria,  the  blastomycetes,  and  the 

hyphomycetes. 

Bacillus  mallei 

Synonyms. — Bacterium  mallei;  Mycobacterium  mallei. 

Diseases  Produced. — Glanders  and  farcy  in  equines;  Rotz; 
morve. 

Loffler  and  Schiitz,  in  1882,  demonstrated  the  presence  of  a 
characteristic  rod  (B.  mallei)  in  the  nasal  discharge  of  a  horse 
affected  with  glanders.  Kitt,  in  4883',  and  Weichselbaum,  in 
1885,  confirmed  these  results  and  added  to  our  knowledge  of  the 
organism. 

Distribution. — Glanders  is  known  in  practically  every 
civilized  country. 

Morphology  and  Staining. — Bacillus  mallei  is  a  short  rod, 
usually  straight,  but  sometimes  somewhat  curved.  The  ends  are 
rounded.  It  is  usually  single,  more  rarely  in  pairs  or  short  chains. 
Involution  forms  are  frequently  produced;  enlarged  cells,  clubbed 
forms,  filaments,  and  even  branching  have  been  observed.  This 
last  fact  has  led  to  the  grouping  of  this  form  with  the  higher  fungi 
by  some  authors.  The  normal  rods  vary  from  0.25  to  0.4  by  1.5  to 
5  p.  The  organism  is  non-motile,  and  does  not  produce  spores 
or  capsules.  It  stains  with  the  ordinary  anilin  dyes,  and  still 
better  with  stains  containing  a  mordant,  such  as  carbol-fuchsin. 

250 


GLANDERS   GROUP 


251 


It  sometimes  shows  some  granular  differentiation  of  the  cytoplasm, 
resembling  the  diphtheria  bacillus.  It  is  not  acid  fast  and  is 
gram-negative . 

Isolation  and  Culture. — Bacillus  mallei  is  rarely  in  pure  cul- 
tures in  the  nasal  discharges,  so  that  for  its  isolation  from  such 
sources  a  special  technic  is  necessary.  It  is  customary  to  inject 
intraperitoneally  a  male  guinea-pig  with  a  small  quantity  of  the 
discharge  from  an  ulcer,  mixed  with  a  little  bouillon  or  physiologic 
salt  solution.  Within  two  to  four  days  the  testicles  swell  and  give 
evidence  of  acute  inflammation.  The  animal  is  then  killed,  a 
testis  removed  and  opened  under  aseptic  conditions,  and  the  con- 
tents of  one  of  the  small  abscesses  or  foci  of  inflammation  removed 
on  a  sterile  platinum  needle  „  ,  ^ 

to  suitable  media. 

Bacillus  mallei  grows 
upon  the  ordinary  culture- 
media,  particularly  upori 
those  that  contain  glycerin, 
upon  blood-serum,  and 
potato.  The  colonies  upon 
agar  and  glycerin-agar 
plates  are  whitish  or  yellow- 
ish, glistening,  usually  cir- 
cular. Upon  the  slanted 
medium  the  colonies  are 
coalescent  and  form  a 
moist,  shining  layer.  In 
bouillon  and  glycerin  bouillon  B.  mallei  produces  an  initial  tur- 
bidity, followed  by  sedimentation;  a  shining  white  pellicle  is 
likewise  formed  when  the  medium  is  not  shaken.  On  blood- 
serum  the  colonies  are  first  discrete,  clear,  yellowish,  viscous, 
hemispheric  drops  which  coalesce  to  form  "a  transparent  layer 
over  the  surface;  this  later  becomes  gray  and  opaque.  Gela- 
tin is  not  liquefied.  The  growth  upon  potato  is  perhaps  the 
most  characteristic.  It  may  be  described  as  forming  within  forty- 
eight  hours  a  yellow,  honey-like,  semitransparent  growth  that 
gradually  becomes  brownish  or  amber  in  tint.  The  potato  itself 
is  tintd  greeenish  or  greenish  brown.  This  reaction  is  not  charac- 


Fig.   106. — Bacillus  mallei  from  glycerin 
agar  (X  1000[  (Frankel  and  Pfeiffer). 


252 


VETERINARY    BACTERIOLOGY 


teristic  if  potatoes  having  too  acid  a  reaction  are  used.  They  may 
be  neutralized  previously  to  inoculation  by  soaking  in  dilute  sodium 
carbonate. 

Physiology. — B.  mallei  is  aerobic  and  facultative  anaerobic. 
Its  optimum  growth  temperature  is  37°,  but  its  growth  limits,  at 
least  in  freshly  isolated  cultures,  are  about  25°  and  42°.  Its 
thermal  death-point  is  55°,  with  five  minutes'  exposure. 

Pathogenesis. — Experimental  Evidence. — There  is  an  abun- 
dance of  evidence  to  prove  that  B.  mallei  is  the  cause  of  glanders. 
All  the  lesions  of  the  disease  may  be  duplicated  by  the  experi- 
mental inoculation  of  pure  cultures  into  laboratory  animals  and 

the  horse.  The  guinea-pig 
is  very  susceptible.  A  sub- 
cutaneous inoculation  is  fol- 
lowed within  a  few  days  by 
local  swelling  and  indura- 
tion, which  soon  ulcerates 
and  discharges  to  the 
surface.  The  disease 
spreads  largely  through  the 
lymph-channels,  and  the 
lymph-nodes  enlarge  and 
suppurate.  Various  meta- 
static  infections  of  the 
joints,  the  lungs,  and  other 
organs  occur.  Death  seems 
to  be  due  to  exhaustion. 

Infection  may  similarly  be  transmitted  to  the  rabbit.  The  horse 
may  be  readily  infected,  as  may  sheep,  goats,  the  cat,  and  the 
dog.  Cattle  and  the  house-rat  do  not  contract  the  disease.  It 
occurs  in  man  through  infection  from  glandered  animals  and 
through  working  with  pure  cultures  in  the  laboratory. 

Character  of  Disease  and  Lesions  Produced. — The  disease  as 
found  in  equines  may  be  either  of  an  acute  or  a  chronic  type. 
The  former  is  commoner  in  the  ass  and  mule,  and  the  latter  in 
the  horse.  The  acute  type  of  disease  is  commonly  ushered  in  with 
a  chill,  there  is  a  mucopurulent  discharge,  and  death  usually  occurs 
in  from  one  to  four  weeks.  The  chronic  type  shows  no  marked 


Fig.  107. — Bacillus  mallei,  in  section 
from  the  spleen  of  a  field-mouse  (Frankel 
and  Pfeiffer). 


GLANDERS   GROUP  253 

characteristics  in  its  early  stages;  the  lymph-nodes  in  various 
parts  of  the  body  become  infected  and  enlarge.  This  may  exist 
for  a  long  period  in  an  animal,  and  may  terminate  finally  in  an 
acute  attack.  The  lesions  in  the  chronic  type  are  generally  present 
on  the  nasal  mucosa,  in  the  lungs,  and  in  the  lymph-glands.  The 
nodular  glanders  of  the  nasal  mucosa  is  the  most  frequent  type. 
The  nodules,  small  at  first,  enlarge  to  the  size  of  a  pea,  then  break 
down,  suppurate,  and  form  chronic  ulcers.  When  healing  of  the 
deeper  ulcers  occurs,  the  scar  resulting  is  quite  characteristic. 
In  the  lungs  lesions  are  almost  invariably  to  be  found ;  these  may  be 
nodular,  or  consist  of  infiltration  of  considerable  areas  of  tissue. 
In  farcy  or  cutaneous  glanders  the  nodules  form  in  the  skin; 
the  lymph-vessels  become  swollen  and  feel  like  a  string  of 
beads  or  a  knotted  cord.  These  nodules  usually  break  through 
to  the  surface  and  ulcerate.  In  man  the  organism  commonly 
gains  entrance  through  abrasions  or  wounds  in  the  skin,  or 
by  inhalation,  and  the  infection  produced  is  practically  always 
fatal. 

Immunity. — No  toxins  have  been  demonstrated  for  Bacillus 
mallei,  although  endotoxins  are  produced.  Agglutinins  are  pres- 
ent in  the  blood-serum  of  normal  animals,  but  in  much  greater 
concentration  in  the  blood  of  infected  animals.  Precipitins 
may  also  be  demonstrated.  Of  the  bactericidal  and  opsonic 
nature  of  sera  less  is  known. 

Active  Immunization. — Immunization  by  the  use  of  suspensions 
of  dead  bacteria  or  their  products  (mallein)  has  been  attempted 
both  in  prophylaxis  and  cure.  Although  some  favorable  results 
have  been  reached,  the  subject  needs  further  study.  No  method 
of  vaccination  or  active  immunization  has  as  yet  been  shown  to  be 
practical  and  successful. 

Passive  Immunization. — The  blood-serum  of  animals,  such  as 
the  ox,  naturally  immune  to  glanders  has  been  claimed  to  possess 
immunizing  power  when  injected  into  smaller  laboratory  animals, 
as  the  rabbit.  No  practical  utilization  has  been  made  of  this,  and 
the  fact  itself  is  in  need  of  further  study. 

Bacteriologic  Diagnosis. — A  presumptive  bacteriologic  diag- 
nosis may  be  made  by  an  examination  of  properly  stained  pus 
or  sections  of  tissue,  and  a  more  positive  diagnosis  by  the  meth- 


254  VETERINARY   BACTERIOLOGY 

ods  of  animal  inoculation,  agglutination,  precipitation,  absorption 
of  complement,  and  by  the  use  of  mallein. 

Examination  of  Pits  and  Tissues. — The  discovery  of  a  gram- 
negative  bacillus  (in  the  pus  or  in  tissues)  having  the  general 
characters  of  the  glanders  bacillus  is  presumptive  evidence  of  its 
presence,  as  there  are  few  organisms  with  which  it  might  be  con- 
fused. Inasmuch  as  the  organism  decolorizes  easily  and  is  gram- 
negative  it  is  difficult  to  demonstrate  satisfactorily  in  tissues. 
The  method  of  Kiihne  is  recommended  as  giving  good  results. 
Carbol-methylene-blue  (methylene-blue,  1.5  gm.;  alcohol,  10  c.c., 
and  5  per  cent,  aqueous  phenol  or  carbolic  acid,  100  c.c.)  is  used  to 
stain  the  sections  one-half  hour;  they  are  then  washed  in  water, 
then  in  very  dilute  hydrochloric  acid  (10  drops  to  500  c.c.  of  water), 
and  quickly  transferred  to  a  solution  of  lithium  carbonate  (8  drops 
of  a  saturated  solution  to  10  c.c.  of  water),  then  to  distilled  water, 
dehydrated  in  absolute  alcohol  containing  a  little  methylene-blue, 
then  cleared  in  anilin  oil.  The  bacteria  should  show  plainly. 

Diagnosis  by  Animal  Inoculation. — A  male  guinea-pig  is  in- 
oculated intraperitoneally  with  a  small  amount  of  the  suspected 
material.  This  should  be  secured  as  free  as  possible  from  other 
organisms  to  obviate  the  possibility  of  the  animal  dying  pre- 
maturely of  peritonitis  or  septicemia.  In  from  two  to  four  days 
the  testes  become  enlarged  and  tender,  the  skin  above  them  is 
reddened  and  shiny.  The  animal,  in  case  of  a  positive  reaction, 
should  be  killed  and  the  contents  of  the  testes  examined  micro- 
scopically to  determine  the  presence  of  a  gram-negative  charac- 
teristic bacillus.  Other  organisms  may  give  the  orchitic  reaction, 
but  they  are  gram-positive,  with  the  exception  of  Bacillus  pyo- 
cyaneus.  A  culture  should  always  be  made  from  the  pus  in  the 
scrotum  to  make  diagnosis  certain.  This  reaction  is  sometimes 
known  as  Strauss'  biologic  test. 

Agglutination  Test  for  Glanders. — The  serum  of  a  normal  horse 
will  frequently  agglutinate  the  B.  mallei  when  in  dilutions  of  1 :  100, 
1 :  500,  rarely  more.  The  serum  from  infected  animals  will  in 
general  give  a  reaction  in  dilutions  of  1 :  500,  and  usually  much 
higher.  The  organisms  used  in  the  agglutination  test  may  be 
either  living  or  dead.  The  latter  are  commonly  used,  as  it  docs 
away  largely  with  danger  of  infection  to  man.  The  bacterial 


GLANDERS   GROUP  255 

suspension  is  prepared  by  removing  the  growth  from  a  young 
culture  on  agar  and  suspending  it  in  physiologic  salt  solution  con- 
taining 0.5  per  cent,  phenol.  This  is  heated  at  70°  for  two  to 
four  hours;  this  kills  the  bacteria,  but  does  not  interfere  with  the 
agglutination  reaction.  Equal  amounts  of  this  suspension  are 
placed  in  a  series  of  small  test-tubes,  and  to  these  are  added  equal 
amounts  of  different  dilutions  of  the  serum  to  be  tested,  and  the 
final  dilutions  of  the  serum  determined.  Dilutions  are  usually 
prepared  1: 100,  1: 200,  1: 400,  1: 500,  1:  800,  1: 1000,  and  up  to 
1 :  8000  or  more.  The  tubes  are  kept  at  37  °  for  from  twenty-four 
to  thirty-six  hours.  A  positive  reaction  is  indicated  by  a  film 
covering  the  entire  bottom  of  the  tube,  a  negative  by  no  pre- 
cipitate or  a  little  sediment  in  the  bottom  of  the  convexity,  not 
forming  a  film.  Normal  blood  frequently  gives  the  reaction  in 
dilutions  as  high  as  1 : 500,  and  usually  in  dilutions  of  1 : 100  or 
less.  The  serum  of  injected  animals  will  commonly  agglutinate  in 
dilutions  of  1 :  800,  1 :  1000,  and  much  higher.  A  positive  reaction 
in  dilutions  of  over  1 :  1000  may  be  considered  diagnostic.  Whether 
or  not  a  positive  reaction  is  accompanied  by  a  complete  clearing 
of  the  test  fluid  depends  upon  the  concentration  of  the  suspension 
and  the  dilution  and  potency  of  the  serum  used.  The  fluid  may 
remain  somewhat  cloudy  in  a  positive  reaction  in  the  higher 
dilutions,  not  all  the  organisms  being  agglutinated.  The  sus- 
pensions of  killed  organisms  may  be  secured  ready  for  use  from 
some  pharmaceutical  houses,  together  with  tubes  and  materials  for 
preparing  the  proper  dilutions.  The  suspension  when  properly 
prepared  and  preserved  in  the  dark  will  keep  for  a  considerable 
time.  The  microscopic  test  for  agglutination  has  not  proved 
practicable,  as  normal  serum  agglutinates  microscopically  in 
high  dilutions.  When  properly  carried  out,  the  macroscopic 
test  is  claimed  by  some  to  be  an  even  better  diagnostic  than 
mallein. 

Konew's  Precipitation  Test  or  the  Ring  Test. — A  solution  of 
glanders  bacillus  prepared  by  adding  10  c.c.  of  an  8  per  cent, 
antiformin  *  solution  to  the  bacilli  washed  from  the  surface  of  a 
forty-eight-hour  slant  agar  culture.  The  bacteria  will  go  into 

!The  composition  of  antiformin  is  given  on  p.  312.  It  is  a  patented 
disinfecting  solution,  and  may  be  purchased  upon  the  market. 


256  VETERINARY    BACTERIOLOGY 

solution  within  two  hours.  It  is  well  to  add  even  more  of  the  or- 
ganism if  it  appears  to  dissolve  rapidly,  as  it  is  desirable  to  get  as 
concentrated  a  solution  as  is  possible.  The  solution  must  then 
be  carefully  neutralized,  preferably  by  the  use  of  5  per  cent,  sul- 
phuric acid.  This  is  then  filtered  through  paper,  then  through 
a  Berkefeld  filter,  to  remove  all  undissolved  bacteria.  The  filtered 
solution  is  termed  "  mallease."  A  test-tube  is  filled  to  a  depth  of 
3  cm.  with  mallease,  and  blood-serum  from  a  suspected  case  is 
introduced  by  means  of  a  pipette.  The  end  of  the  pipette  should 
be  passed  through  the  layer  of  mallease  and  should  rest  against  the 
bottom  of  the  tube  before  the  serum  is  allowed  to  flow.  A  quantity 
of  serum  about  equal  to  the  mallease  is  introduced.  The  pipette  is 
withdrawn  quickly  and  carefully  to  prevent  any  mixture  of  the 
two  liquids.  The  serum  has  a  higher  specific  gravity  and  remains 
at  the  bottom,  with  the  mallease  as  a  distinct  superficial  layer. 
If  the  serum  is  from  an  animal  free  from  the  disease,  there  will  be 
no  reaction.  A  positive  diagnosis  of  glanders  is  indicated  by  a 
white  cloudiness  that  appears  along  the  line  separating  the  two 
liquids.  This  is  due  apparently  to  precipitation  by  the  specific 
precipitins  formed  in  the  serum.  In  acute  or  well-marked  cases 
the  reaction  occurs  almost  immediately,  and  usually  in  all  cases 
within  fifteen  minutes.  The  test  is  too  recent  to  speak  authorita- 
tively of  its  reliability,  but  the  reports  thus  far  seem  to  indicate 
that  it  is  far  more  reliable  and  more  easily  carried  out  than  any  of 
the  other  glanders  tests. 

Fixation  of  Complement  Test. — Schlitz  and  Schubert1  have 
described  a  satisfactory  method  of  adapting  Wassermann's  syphilis 
test  by  fixation  of  complement  to  the  diagnosis  of  glanders.  Mohler 
and  Eichhorn2  have  tested  out  the  method  and  found  it  highly 
satisfactory.  They  prepare  hemolytic  amboceptor  for  sheep 
erythrocytes  by  injecting  the  washed  red  blood-cells  intraperi- 
toneally  into  a  rabbit.  The  corpuscles  are  suspended  in  an  equal 
bulk  of  physiologic  salt  solution,  and  14,  20,  and  24  cubic  centi- 
meters are  injected  at  intervals  of  seven  days.  The  serum  from 
the  blood  of  the  rabbit  may  be  used  in  five  or  six  days  after  the  last 
injection.  It  must  be  inactivated  by  heating  to  56°  for  thirty 

1  Arch.  f.  Wiss.  u.  prakt.  Ticrlx -ilkunde,  Band  35,  pp.  44-83,  1909. 

2  Bull.  136,  Bureau  An.  Ind.  U.  S.  Dept.  of  Agriculture. 


GLANDERS   GROUP  257 

• 

minutes  before  it  can  be  used.  Fresh  guinea-pig  serum  is  used  as 
complement.  The  antigen  used  is  an  extract  of  glanders  bacilli 
prepared  from  the  growth  on  slant  glycerin-agar  tubes.  The 
growth  is  washed  off  with  physiologic  salt  solution  and  heated  to 
60°  for  four  hours  to  kill  the  bacteria.  The  suspension  of  organ- 
isms is  then  placed  in  flasks  and  shaken  in  a  shaking  apparatus  for 
four  days.  It  is  then  centrifuged,  the  clear  liquid  removed,  and 
10  per  cent,  of  a  5  per  cent,  solution  of  phenol  added.  This  anti- 
gen may  be  preserved  without  material  deterioration  for  several 
months  if  kept  in  a  cool,  dark  place. 

It  is  necessary  to  titrate  the  rabbit  serum  and  likewise  the 
antigen  in  order  to  determine  the  amounts  most  suitable  for  carry- 
ing out  the  test.  For  each  set  of  determinations  of  diagnosis  fresh 
guinea-pig  serum  must  be  used.  Blood-serum  from  the  animal 
that  is  suspected  of  having  glanders  must  be  inactivated  by  heat- 
ing to  58°  for  thirty  minutes.  The  materials  necessary  for  the 
test  are — 

1.  Washed  sheep  corpuscles,  5  per  cent,  suspension  (antigen  1). 

2.  Inactivated  serum  from  rabbit  immunized  against  1  (am- 
boceptor  1). 

3.  Fresh  guinea-pig  serum  (complement). 

4.  Extract  of  glanders  bacilli  (antigen  2). 

5.  Inactivated  serum  from  suspected  animal  (amboceptor  2). 
The  test  is  carried  out  in  test-tubes.     In  tubes  1  and  2  there  is 

placed  0.1  c.c.  of  the  serum  (No.  5,  above),  and  in  tubes  3  and  4, 
0.2  c.c.  of  the  same.  One  c.c.  of  the  established  dilution  of  glanders 
bacilli  (No.  4,  above)  is  then  added  to  tubes  1  and  3.  To  each  tube 
is  then  added  1  c.c.  of  the  dilution  of  fresh  guinea-pig  serum  that 
has  been  established  by  preliminary  test.  Each  tube  is  now  made 
up  to  3  c.c.  with  physiologic  salt  solution.  They  are  then  placed 
in  the  thermostat  at  37°  for  an  hour.  They  are  then  removed  and 
to  each  tube  is  added  1  c.c.  of  the  previously  standardized  rabbit 
serum  (No.  2,  above)  and  1  c.c.  of  the  sheep  corpuscles  (No.  1, 
above).  The  tubes  are  shaken  and  incubated  for  ten  hours. 
A  positive  diagnosis  is  indicated  by  lack  of  hemolysis  in  tubes  1  and 
3  and  complete  hemolysis  in  tubes  2  and  4.  Checks  must  be  made 
to  determine  the  hemolytic  activity  of  each  of  the  above  con- 
stituents independently. 

17 


258  VETERINARY   BACTERIOLOGY 

This  method  is  essentially  a  laboratory  one  and  quite  impracti- 
cable for  field  work.  There  seems  to  be  no  reason  why  blood 
samples  or,  better,  serum  samples  from  suspected  cases  should  not 
be  sent  to  properly  equipped  laboratories  for  diagnosis  and  report. 
The  method  apparently  is  capable  of  giving  good  results,  and  seems 
to  be  more  accurate  than  the  mallein  test. 

Mallein  Test  for  Glanders. — Mallein  is  a  suspension  of  killed 
B.  mallei,  together  with  the  products  of  its  autolytic  disintegration. 
What  the  active  principles  in  bringing  about  the  characteristic 
reaction  in  a  glandered  horse  may  be  is  not  known.  Probably 
they  are  the  soluble  bacterial  proteins,  possibly  true  endotoxins. 
The  various  laboratories  use  different  methods  of  preparing  mal- 
lein. The  most  important  of  these  are  worthy  of  note. 

The  mallein  of  Roux  is  prepared  by  the  Pasteur  Institute  as 
follows:  The  virulence  of  the  B.  mallei  used  is  increased  by  pas- 
sage through  rabbits,  and  is  such  that  mice  and  rabbits  are  killed 
in  less  than  thirty  hours  by  intravenous  injections.  Flasks 
containing  250  c.c.  of  glycerin  bouillon  are  inoculated  and  in- 
cubated a  month  at  35°.  The  cultures  are  killed  by  exposure 
to  a  temperature  of  100°  for  thirty  minutes  in  an  autoclave,  then 
evaporated  to  one-tenth  the  volume,  and  filtered  through  filter- 
paper  ("  papier  Chardin  ").  The  final  product  is  a  dark-brown, 
syrupy  liquid,  containing  50  per  cent,  glycerin.  For  use  this  is 
mixed  with  nine  times  its  volume  of  0.5  per  cent,  carbolic  acid. 
The  diagnostic  dose  is  2.5  c.c.  of  this  dilution. 

The  mallein  of  Vladimiroff,  used  in  the  Russian  Empire,  is 
prepared  by  inoculating  a  considerable  number  of  flasks,  each 
containing  600  to  800  c.c.  of  beef  broth,  with  a  vigorous  culture 
of  B.  mallei,  and  incubating  for  eight  months  at  37°.  The  flasks 
are  shaken  from  time  to  time  to  cause  ohe  shiny,  gray-white  pel- 
licle which  forms  to  sink  to  the  bottom.  The  culture  is  then 
examined  for  purity,  sterilized  in  the  autoclave  at  110°,  and  filtered. 
This  is  concentrated  and  again  diluted  until  the  diagnostic  dose  for 
the  horse  is  1  c.c. 

The  mallein  or  morvin  of  Babes  is  prepared  by  inoculating 
potato  paste  with  B.  mallei,  and  incubating  six  weeks.  It  then 
is  heated  at  68°  for  three  and  one-half  hours,  emulsified  with  water, 
filtered  through  a  Witt  filter,  and  precipitated  with  alcohol.  This 


GLANDERS   GROUP  259 

precipitate  is  washed  in  alcohol,  then  in  ether,  and  dried.  The 
diagnostic  dose  is  0.02  to  0.03  gm.  It  is  prepared  for  injection  by 
dissolving  in  a  mixture  of  glycerin  and  water. 

The  malleinum  siccum,  or  dried  mallein,  of  Foth  is  prepared 
by  growing  B.  mallei  in  4.5  per  cent,  glycerin  broth.  The  cultures 
used  are  rendered  virulent  by  passage  through  cats,  guinea-pigs, 
and  field-mice.  The  material  is  incubated  at  37.7°  for  three 
weeks.  It  is  concentrated,  and  the  organism  killed  by  evapora- 
tion at  a  constant  temperature  of  76°  to  80°  to  one-tenth  of  its 
former  volume.  This  is  filtered  and  poured  into  absolute  alcohol, 
in  which  a  precipitate  immediately  forms.  This  precipitate  is 
washed  in  alcohol  and  dried  in  a  desiccator.  The  final  product 
is  a  white  powder  which  readily  dissolves  in  water.  The  diag- 
nostic dose  for  the  horse  is  0.045  to  0.05  gm. 

The  mallein  prepared  in  the  laboratories  of  the  Bureau  of  Animal 
Industry  consists  of  glycerinated  broth  in  which  the  B.  mallei 
has  grown  four  to  five  months,  has  been  heated,  concentrated,  and 
filtered.  It  is  diluted  by  the  addition  of  one-half  its  volume  of 
glycerin  and  one  and  one-half  times  its  volume  of  1  per  cent, 
phenol.  The  diagnostic  dose  is  1  c.c. 

No  practicable  method  of  standardizing  mallein  has  been 
worked  out  other  than  trial  upon  a  considerable  number  of  healthy 
and  infected  animals.  The  variations  in  the  methods  of  produc- 
tion of  mallein  given  above  are  due  to  a  desire  to  secure  a  very 
uniform  product. 

Mallein,  when  injected  in  the  correct  diagnostic  dose,  some- 
times causes  in  a  healthy  animal  a  slight  fever  reaction  of  short 
duration,  with  frequently  a  transitory  swelling  at  the  point  of 
injection.  When  injected  into  a  glandered  animal,  the  tempera- 
ture begins  to  rise  in  six  to  eight  hours.  At  the  site  of  injection 
there  is  developed  a  swelling,  painful,  hot,  and  of  considerable  size, 
and  extending  along  the  lymphatics  for  some  distance.  It  persists 
several  days,  and  disappears  in  a  week  or  ten  days.  Constitu- 
tional symptoms  of  the  reaction,  such  as  dejection  of  the  patient, 
lusterless  coat,  anxious  expression,  impaired  appetite,  and  hurried 
respiration  are  usually  valuable  aids  in  recognizing  a  reaction. 

When  properly  carried  out,  the  mallein  test  is  valuable  as  a 
diagnostic  method  for  glanders. 


260  VETERINARY   BACTERIOLOGY 

Transmission. — The  disease  is  transmitted  from  one  animal  to 
another  through  infected  food,  mangers,  drinking  troughs,  etc.; 
rarely  through  wounds  or  skin  abrasions.  Veterinarians  and  horse- 
men sometimes  become  infected  through  the  skin,  rarely  by  in- 
halation. 

Bacilli  of  Setter,  Babes,  and  Kutscher' 

Organisms  morphologically  similar  to  the  preceding  have 
been  isolated  from  pus  by  Selter  and  by  Babes,  and  from  the  nos- 
trils of  a  healthy  horse  by  Kutscher.  They  may  be  differen- 
tiated readily  by  their  lack  of  pathogenesis,  and  would  rarely, 
if  ever,  lead  to  mistakes  in  diagnosis. 


CHAPTER  XXVIII 

INTESTINAL  OR  COLON-TYPHOID    GROUP.    WATER    ANALYSIS 

THE  organisms  belonging  to  this  group  may  be  characterized 
as  plump,  gram-negative  rods,  frequently  though  not  always 
motile;  they  produce  no  spores,  do  not  liquefy  gelatin,  and  in  most 
cases  ferment  certain  sugars,  with  acid-  and  sometimes  gas- 
production.  The  group  contains  many  undoubted  species  that 
may  be  easily  differentiated,  but  there  are  many  intergrading 
types  and  forms  showing  similar  morphologic  and  cultural  char- 
acters, but  differing  considerably  in  kind  and  in  degree  of  virulence. 
These  latter  make  a  systematic  presentation  of  the  group  as  a 
whole  difficult.  Exactly  what  amount  of  difference  is  necessary 
to  constitute  a  distinct  species  is  always  a  difficult  problem,  but 
nowhere  more  so  than  with  these  forms.  The  group  name  is 
given  because  of  their  prominence  in  the  intestinal  flora  in  disease 
and  health  in  both  man  and  animals.  They  are,  therefore,  abun- 
dant in  sewage  and  water  contaminated  thereby,  and  in  soil,  par- 
ticularly that  which  has  received  additions  of  barn-yard  manure. 
They  are  uncommon  in  virgin  soil  and  in  uncontaminated  water. 

The  members  of  this  group  are  divided,  for  convenience  in 
study,  into  three  subgroups.  This  arrangement  seems  to  repre- 
sent evident  relationships.  The  fermentative  powers  of  the 
organisms  are  used  as  a  basis  upon  which  to  make  the  groupings. 
The  first  of  these  is  known  as  the  colon  bacillus  subgroup,  the 
second  as  the  intermediate,  hog-cholera,  or  enteritidis  subgroup, 
and  the  third  as  the  typhoid-dysentery  subgroup.  The  principal 
points  of  difference  between  these  subgroups  may  be  summarized 
in  the  following  chart,  giving  the  fermentation  reactions  in  dex- 
trose and  lactose  broth. 

Subgroup  I.  Subgroup  II.  Subgroup  III. 

Colon  subgroup.          Intermediate  subgroup.  Typhoid-dysentery   sub- 
group. 
Acid.  Gas.  Acid.  Gas.  Acid.  Gas. 

Dextrose -*-       4-    Dextrose 4-        4-     Dextrose ±       — 

Lactose 4-       4-    Lactose - .  —       —     Lactose —       — 

261 


262  VETERINARY    BACTERIOLOGY 

The  differences  may  be  summarized  as  follows:  the  organisms 
of  subgroup  I.  ferment  both  dextrose  and  lactose,  with  formation 
of  both  acid  and  gas;  those  of  subgroup  II.  form  acid  and  gas  from 
dextrose,  but  not  from  lactose;  and  those  of  subgroup  III.  may  or 
may  not  form  acid  from  dextrose,  but  never  from  lactose,  and  gas 
from  neither  of  the  sugars. 

The  fermentations  of  other  carbohydrates  and  related  com- 
pounds are  used  to  differentiate  species  and  varieties  from  each 
other.  A  few  can  be  satisfactorily  differentiated  only  by  the  ag- 
glutination reaction.  For  a  study  of  the  fermentative  power  of 
the  organisms  1  per  cent,  solutions  of  the  sugars  to  be  studied  are 
made  in  sugar-free  broth  and  placed  in  fermentation  tubes,  and 
sterilized  by  the  discontinuous  process  to  prevent  decomposition. 
Those  organisms  which  produce  gas  grow  in  both  the  open  and 
closed  arm,  as  do  those  which  produce  acid,  and  those  which  do 
not  ferment  the  sugar  are  usually  confined  to  the  open  arm. 
The  composition  of  the  gas,  that  is,  the  relative  proportion  of 
CO2  and  H2,  is  also  of  diagnostic  value. 

The  organisms  to  be  considered  belonging  to  the  colon  sub- 
group are  Bacillus  coli,  B.  lactis  aerogenes,  and  B.  pneumonia?. 
Those  of  the  intermediate  group,  Bacillus  enteritidis,  B.  cholera 
suis,  B.  psittacosis,  B.  paratyphosus,  B.  typhi  murium,  bacillus  of 
Danysz,  and  B.  pullorum.  The  most  important  forms  of  the 
third  subgroup  are  Bacillus  typhosus,  B.  dysenteries,  and  B. 
fceqalis  alkaligenes. 

The  growth  reactions  of  the  various  members  of  the  intestinal 
group,  and  more  particularly  of  the  colon  subgroup,  are  of  con- 
siderable sanitary  significance,  as  they  furnish  our  most  efficient 
means  of  securing  evidence  of  the  suitability  of  water  for  drinking 
purposes,  and  of  its  contamination  by  sewage.  The  topics  of 
.water  analysis,  sewage  disposal,  and  water  purification  are  so 
intimately  connected  with  the  discussion  of  the  members  of  the 
intestinal  group  that  they  are  included  in  the  same  chapter. 

SUBGROUP  I— COLON  SUBGROUP 

Bacillus  coli 

Synonyms. ^Bacillus  coli  communis;  B.  neapolitanus ;  B. 
pyogenes  fcetidus ;  Bacterium  coli  (commune);  colon  bacillus. 


INTESTINAL   OR   COLON-TYPHOID   GROUP 


263 


Emmerich,  in  1885,  isolated  an  organism,  which  he  named 
Bacillus  neapolitanus,  from  the  feces.  of  patients  suffering  from 
Asiatic  cholera.  Escherich,  in  1886,  isolated  a  similar  organism, 
which  he  termed  Bacterium  coli  commune,  from  normal  feces. 
Since  that  time  the  organism  has  been  found  to  be  constantly 
present  in  the  intestines  of  man,  most  animals,  and  even  some 
birds.  The  question  of  its  occurrence  in  nature  independent  of 
fecal  contamination  is  a  moot  one.  That  it  may  maintain  a  sapro- 
phytic  existence  outside  the  body  for  some  time  seems  to  be  well 
established,  but  the  evidence  that  it  does  not  usually  long  so  main- 
tain itself  is  increasing.  Examination  of  water  for  the  presence  of 
B.  coli  to  determine  its  potability  is  quite  universally  practised, 
and  when  properly  interpreted, 
has  led  to  valuable  results. 
The  presence  of  B.  coli  in  water 
in  any  considerable  numbers  is 
sufficient  to  condemn  it  for 
drinking  purposes,  not  because 
of  a  pathogenic  property  of  this 
organism,  but  simply  because 
it  indicates  contamination  with 
surface  wash  or  with  sewage. 

Morphology  and  Staining. — 
The  B.  coli  is  a  rod,  varying 
from  0.4  to  0.7  by  2  to  4  u, 
sometimes  shorter  and  almost 
coccus-like,  with  rounded  ends, 

usually  single,  but  occasionally  in  short  chains.  It  does  not 
produce  spores  or  capsules.  It  is  rather  sluggishly  motile,  at 
least  in  young  cultures,  usually  with  2  to  8  flagella,  rarely  more. 
It  stains  readily  with  the  ordinary  anilin  dyes,  sometimes  show- 
ing some  vacuolization  and  polar  granules.  It  is  gram-negative. 

Isolation  and  Culture. — B.  coli  may  be  readily  isolated  from 
feces  or  sewage  by  plating  the  material  in  various  dilutions  in 
litmus-lactose  agar,  and  incubating  at  blood-heat.  The  colonies 
of  B.  coli  appear  surrounded  by  a  zone  of  red,  due  to  the  formation 
of  acids  from  the  lactose.  The  colonies  must  be  differentiated 
from  those  of  the  organism  next  to  be  described.  Upon  gelatin 


Fig.  108.— Bacillus  coli,  stained 
preparation  from  a  twenty-four-hour 
agar  slant  (X  650)  (Heim). 


264 


VETERINARY   BACTERIOLOGY 


plates  the  colonies  are  moist,  grayish  white,  opaque,  becoming 
darker  and  more  coarsely  granular.  Gelatin  is  not  liquefied. 
Stab  cultures  in  gelatin  show  a  filiform  growth  along  the  line  of 
puncture,  and  a  spreading  growth  at  the  surface.  The  agar  cul- 
tures resemble  those  on  gelatin.  Bouillon  is  quickly  clouded, 
sometimes  with  formation  of  a  pellicle.  On  potato  a  moist, 
spreading  growth  occurs,  and  the  potato  is  darkened.  Milk  is 

coagulated  by  the  forma- 
tion of  acids;  the  curd 
shrinks,  but  is  -not 
digested. 

Physiology. — B.  coli 
is  aerobic  and  facultative 
anaerobic.  Its  optimum 
growth  temperature  is 
37°,  but  growth  is 
luxuriant  at  room-tem- 
perature and  even  below. 
The  thermal  death-point 
is  60°  for  fifteen  min- 
utes. Many  carbohy- 
drates are  fermented, 
with  production  of  acid 
and  gas.  Among  these 

are  dextrose,  lactose,  and  maltose,  and,  in  about  half  the 
strains  isolated,  saccharose.  The  gas  formula  from  dextrose  is 

H          2 

approximately  ^-  >  <  j.  Indol  is  produced  in  Dunham's  solu- 
tion. Peptonizing  and  proteolytic  enzymes  have  not  been  demon- 
strated. 

Pathogenesis. — The  following  quotation  from  Jordan  epitom- 
izes our  present  estimate  of  the  pathogenicity  of  B.  coli:  "  The 
common  occurrence  of  agonal  or  postmortem  invasion  of  the  body 
by  the  colon  bacillus  tends  to  diminish  the  value  of  the  supposed 
evidence  derived  from  finding  the  colon  bacillus  in  the  internal 
organs  after  death,  and  there  can  be  no  doubt  that  the  role  in 
human  pathology  assigned  to  the  colon  bacillus  by  some  inves- 
tigators, notably  certain  French  bacteriologists,  has  been  greatly 


Fig.  109. — Bacillus  coli  showing  the  flagella 
(Migula). 


INTESTINAL   OR   COLON-TYPHOID   GROUP  265 

exaggerated.  Failure  to  distinguish  between  the  true  colon  group 
and  the  group  of  meat-poisoning  bacilli  is  doubtless  responsible 
for  some  of  the  statements  attributing  pronounced  pathogenic 
properties  to  B.  coli.  The  frequent  ascription  of  various  inflam- 
matory processes,  particularly  those  occurring  in  the  appendix 
and  peritoneum,  to  the  unaided  activities  of  B.  coli,  appears  to  be 
without  sufficient  justification.  Many  of  the  cases  reported  rest 
on  the  evidence  derived  from  simple  aerobic  cultivation,  and  the 
possible  concurrence  of  anaerobic  or  other  organisms  not  growing 
by  ordinary  methods  has  not  been  excluded."  The  preceding 
was  written  with  pathogenesis  for  the  human  body  in  mind,  but 
the  conclusions  are  even  more  true  with  reference  to  its  patho- 
genesis for  animals.  Many  diseases  in  domestic  animals  have 
been  ascribed  to  infection  with  varieties  of  B.  coli  from  insufficient 
evidence.  It  has  been  shown  that  even  in  the  normal  body  colon 
bacilli  sometimes  escape  from  the  intestines,  and  are  to  be  found 
in  the  mesenteric  lymph-nodes,  and  occasionally  in  some  of  the 
other  internal  organs. 

Experimental  Evidence  of  Pathogenesis. — The  intraperitoneal 
injection  of  broth  cultures  of  B.  coli  into  the  guinea-pig  results 
in  the  death  of  the  animal,  usually  within  three  days.  Animal 
experimentation  has  demonstrated  quite  conclusively  that  there 
are  considerable  differences  in  virulence  of  the  colon  bacillus  iso- 
lated from  different  animals  or  from  the  same  animal  at  different 
times. 

Character  of  Lesions  and  Disease  Produced. — B.  coli  has  been 
isolated  from  suppurations  in  pure  culture.  In  man  it  is  known 
occasionally  to  invade  the  gall-bladder,  and  is  a  common  cause  of 
cholecystitis.  It  may  serve  as  a  nucleus  for  gall-stones,  and  is 
probably  instrumental  in  their  formation  by  the  precipitation  of 
cholesterin.  Inflammation  of  the  ureters  and  of  the  urinary 
bladder  is  commonly  caused  in  man  by  organisms  that  cannot  be 
differentiated  from  typical  B.  coli.  It  has  been  reported  as  the 
cause  of  calf  diarrhea  or  white  scours,  and  from  malignant  catarrh 
in  cattle.  It  is  possible  that  these  organisms  are  mere  closely 
related  to  the  B.  enteritidis.  Usually  the  colon  bacillus  does  not 
give  rise  to  putrefactive  products,  and  must  be  regarded  as  a 
harmless  or  even  useful  commensal. 


266  VETERINARY   BACTERIOLOGY 

White  scours,  or  diarrhea  in  calves,  is  the  most  important  of 
the  diseases  of  animals  that  have  been  attributed  to  B.  coli.  Differ- 
ent strains  showing  differences  in  agglutination  have  been  isolated 
from  various  outbreaks.  Moore  and  Nocard  believe  that  the 
organism  enters  the  body  soon  after  birth  through  the  ruptured 
umbilical  cord  coming  in  contact  with  fecal  material.  Investiga- 
tors are  by  no  means  in  accord  in  attributing  this  disease  to  B.  coli. 
The  etiologic  relationship  of  this  organism  cannot  be  considered 
as  settled. 

Immunity. — A  considerable  degree  of  immunity  to  B.  coli 
may  be  induced  by  injections  of  cultures,  killed  or  living.  Ag- 
glutinins  are  present  in  normal  serum,  but  may  be  greatly  in- 
creased by  systematic  immunization.  Specific  precipitins  for 
the  bacterial  proteins  are  present  in  the  immune  serum.  Opsonins 
are  present  in  normal  serum.  The  body  has  naturally  a  high 
degree  of  immunity  against  the  B.  coli.  This  may  be  accounted 
for  by  the  presence  of  B.  coli  in  the  intestines  and  the  continued 
opportunity  for  infection. 

Bacteriologic  Diagnosis. — The  isolation  of  the  characteristic 
colonies  upon  litmus-lactose  agar  is  the  simplest  and  quickest 
method  of  determining  the  presence  of  B.  coli.  Methods  of 
recognition  and  isolation  from  water  will  be  discussed  at  greater 
length  under  that  heading. 

Bacillus  lactis  aerogenes 

Synonyms. — Bacterium  aerogenes;  Bacillus  pyogenes. 

Escherich,  in  1885,  described  an  organism  which  he  isolated 
from  sour  milk  as  B.  lactis  aerogenes.  It  has  since  been  found 
repeatedly  in  the  intestinal  contents  of  man  and  animals.  It  is 
sometimes  more  abundant  than  Bacillus  coli  itself. 

Morphology  and  Staining. — Bacillus  lactis  aerogenes  differs 
morphologically  from  the  B.  coli  principally  by  the  lack  of  flagella 
and  in  the  ability  to  produce  capsules  when  grown  in  milk. 

Isolation  and  Culture. — This  organism  may  be  isolated  in  the 
same  manner  as  B.  coli  upon  litmus-lactose  agar.  The  colonies 
upon  agar  and  gelatin  are  larger,  thicker,  and  more  slimy  than  those 
of  the  colon  bacillus.  Milk  is  curdled  more  rapidly.  In  gelatin 
stabs  the  growth  along  the  streaks  is  filiform;  that  at  the  surface  is 


INTESTINAL   OR   COLON-TYPHOID    GROUP  267 

thick,  convex,  and  circumscribed.  The  whole  stab  culture  is 
frequently  described  as  "  nail  like." 

Physiology. — In  most  respects  this  organism  resembles  B.  coli. 
It  ferments  dextrose,  lactose,  and  saccharose,  with  production 
of  both  acid  and  gas.  It  also  ferments  starch,  notably  by  the  trans- 
formation of  the  starch  into  dextrose  by  an  amylolytic  enzyme. 
Ihdol  is  produced  in  Dunham's  solution. 

Pathogenesis. — This  organism  is  not  known  to  possess  patho- 
genic powers.  It  is  of  interest  principally  because  of  its  close 
relationship  to  B.  coli.  and  association  with  it.  In  making  water 
examinations  no  distinction  is  ordinarily  made  between  B.  coli 
and  B.  lactis  airogenes,  inasmuch  as  they  resemble  each  other  so 
closely  and  come  from  the  same  sources. 

Bacillus  pneumonias 

Synonyms. — Bacterium  pneumonice;  B.  capsulatus  mucosus] 
pneumobacillus;  pneumococcus  of  Friedlander. 

Friedlander,  in  1883,  discovered  this  organism  in  the  sputum 
from  a  case  of  croupous  pneumonia,  and  it  was  believed  by  him 
to  be  the  cause  of  the  disease. 
He  failed  to  discover  the  real 
cause  of   pneumonia   because 
the  pneumococcus  of  Frankel  *  ^ 

does   not   grow   readily  upon       &  ^  ,    ^ 

plate  cultures  prepared  by  the 
method  used.  It  has  since 
been  found  repeatedly  in 
normal  saliva,  and  is  still 
believed  to  be  an  occasional 
cause  of  pneumonia.  It  some- 
times is  found  in  the  feces  and  '*•» 
in  sewage.  fig.  HO.— Bacillus  pneumonice,  show- 

Morphology  and   Staining  ing  capsules  (Gunther). 

— The  organism   as  it  occurs 

in  the  sputum  is  sometimes  so  short  as  to  resemble  a  coccus. 
Usually  it  is  single,  rarely  in  chains.  It  is  surrounded  by  a 
capsule  in  sputum  and  in  milk.  It  is  non-motile.  It  resembles 
the  preceding  organism  closely  in  all  other  respects. 


268  VETERINARY   BACTERIOLOGY 

Isolation  and  Culture. — Isolation  is  accomplished  by  plating 
upon  gelatin.  Growth  upon  most  media  resembles  that  of  the 
B.  lactis  aerogenes.  Milk  is  not  coagulated,  although  litmus  milk 
is  reddened. 

Physiology. — The  organism  shows  markedly  less  fermentative 
power  than  B.  lactis  aerogenes,  but  otherwise  closely  resembles  it. 
Dextrose,  lactose,  and  saccharose  are  all  fermented,  but  usually 
not  vigorously.  Growth  occurs  at  blood-heat,  but  the  organism 
develops  well  at  room-temperature.  The  thermal  death-point  is 
about  56°.  Indol  is  produced. 

Pathogenesis. — B.  pneumonias  has  a  very  low  virulence — only 
exceptionally  will  it  infect  any  of  the  lower  animals.  It  has  been 
isolated  in  pure  culture  from  the  vegetations  upon  the  heart 
valve  in  endocarditis,  from  otitis  media,  and  occasionally  it  is 
believed  to  cause  catarrhal  or  lobular  pneumonia.  It  is  noted 
here  simply  because  of  the  possibility  of  isolation  in  various  infec- 
tions, and  because  of  its  obvious  relationship  to  the  preceding 
organisms  of  the  group. 

SUBGROUP  n— INTERMEDIATE,  HOG-CHOLERA,  OR  ENTERITIDIS 

SUBGROUP 

The  classification  and  relationships  of  the  organisms  belonging 
to  this  subgroup  are  much  confused  at  present.  Whether  or  not 
the  various  forms  described  are  all  distinct  species  is  doubtful. 
The  most  important  will  be  discussed  under  the  names  by  which 
they  are  commonly  known,  but  this  uncertainty  as  to  correct 
grouping  must  constantly  be  borne  in  mind. 

Bacillus  cnteritidis 

Synonym. — Bacillus  of  Gartner. 

Diseases  Produced. — Meat-poisoning  and  enteritis  in  man  and 
in  cattle. 

Gartner,  in  1888,  studied  an  outbreak  of  meat-poisoning  in  a 
village  in  Saxony,  and  isolated  from  a  fatal  case  and  from  the 
uncooked  flesh  of  a  cow  the  organism  now  known  as  Bacillus 
enteritidis.  This  organism  has  since  that  time  been  found  in 
similar  outbreaks  of  meat-poisoning  and  associated  with  certain 


INTESTINAL   OR    COLON-TYPHOID   GROUP 


269 


infections  in  cattle.     The  organism  has  been  found  in  meat  or 
so-called  ptomain-poisoning  in  the  United  States. 

Morphology  and  Staining. — Bacillus  enteritidis  resembles  B. 
coli  morphologically.  The  organism  is  short  and  thick,  sometimes 
with  a  thin  capsule,  motile  by  means  of  numerous  or  few  flagella. 
It  does  not  produce  spores.  It  stains  well  or  irregularly  with  the 
anilin  dyes,  and  is  gram-negative. 

Isolation  and  Culture. — The  organism  has  been  isolated  directly 
from  the  blood-stream  and  the  spleen,  and  from  the  intestinal  con- 
tents by  plate  cultures.  The  cultural  characters  as  reported  vary 
with  different  authors,  probably  because  different  strains  were 
studied.  Colonies  upon  gelatin  and  agar  resemble  those  of  B.  coli. 
Bouillon  is  clouded,  a  delicate 
pellicle  may  form,  and  in  a  few 
days  a  whitish  sediment  collects. 
A  yellowish,  glistening  layer 
forms  on  potato,  frequently  turn- 
ing brownish  with  age.  Growth 
in  milk  seems  to  vary  with  the  or- 
ganism studied.  Some  have  been 
described  as  coagulating  milk, 
but  the  typical  form  does  not, 
although  a  slow  proteolysis  may 
take  place  without  coagulation. 

Physiology. — B.  enteritidis  is 
aerobic  and  facultative  an- 
aerobic. Its  optimum  growth  temperature  is  between  30 


Fig.  111.— Bacillus  enteritidis  (Kolle 
and  Wassermann). 


and 


40°,  but  it  grows  well  at  room-temperature  also.  The  thermal 
death-point,  as  determined  by  Mohler  and  Buckley,  is  58°  for 
twelve  minutes.  Dextrose  is  fermented,  with  production  of  acid 
and  gas.  Lactose  is  not  fermented  by  the  typical  strains,  although 
some  strains  have  been  reported  by  a  few  investigators  to  ferment 
this  sugar,  and  the  statement  is  commonly  current  in  texts.  Indol 
is  not  produced.  Gelatin  is  not  liquefied. 

Pathogenesis. — Experimental  Evidence. — B.  enteritidis  is  patho- 
genic for  the  guinea-pig,  mouse,  and  pigeon,  but  not  for  the 
cat.  The  guinea-pig  may  be  fatally  infected  by  intraperitoneal 
or  subcutaneous  injections  and  by  ingestion.  The  same  is  true 


270  VETERINARY  BACTERIOLOGY 

of  the  rabbit.  Mohler  and  Buckley  produced  a  fatal  infection  in 
young  house-rats,  while  other  authors  report  the  rat  as  immune. 
The  same  is  true  of  the  dog,  though  this  animal  is  relatively  resist- 
ant. Chickens  are  immune.  Sheep  are  readily  infected.  The 
hog  succumbs  to  intravenous  injection,  as  well  as  through  feeding. 

Type  of  Disease  and  Lesions  Produced. — In  man  the  infection 
is  marked  by  a  severe  enteritis  and  enlargement  of  the  lymph- 
follicles  and  Peyer's  patches,  and  by  small  hemorrhages.  The 
mortality  in  infections  is  low — probably  less  than  5  per  cent. 

The  infection  in  cattle,  as  observed  by  Mohler  and  Buckley, 
was  characterized  by  degeneration  of  the  heart  muscle  (frequently 
fatty)  and  hemorrhages  therein;  in  the  liver  parenchymatous 
degeneration  was  accompanied  by  localized  hemorrhagic  extravasa- 
tions; the  spleen  was  enlarged  and  hemorrhagic  and  the  lesions 
of  acute  enteritis,  with  necrosis  of  the  epithelium,  were  evident. 

Immunity. — The  so-called  "  toxin  "of  the  B.  enteritidis  is  prob- 
ably an  unusually  soluble  and  potent  endotoxin.  It  differs  from 
true  toxins  in  being  exceptionally  heat  resistant.  Meat  which 
has  been  quite  thoroughly  cooked  is  sometimes  found  capable  of 
giving  rise  to  toxic  symptoms  when  ingested.  The  bacteria-free 
nitrates  from  bouillon  cultures  and  cultures  in  which  the  organisms 
have  been  killed  by  heat  will  kill  guinea-pigs  when  injected  in 
suitable  quantities.  It  is  probable  that  this  endotoxin  is  respon- 
sible for  the  quick  development  of  symptoms  in  those  who  are 
poisoned  by  eating  infected  food.  Specific  agglutinins  are  devel- 
oped in  the  blood  of  infected  individuals,  as  are  also  coagglutinins 
for  other  members  of  the  intestinal  group.  Some  differences  in 
agglutinability  of  the  different  strains  isolated  have  been  noted. 
It  has  been  proposed  that  meat  may  be  tested  for  the  presence  of 
B.  enteritidis  by  expressing  the  juice  and  determining  its  agglutin- 
ating power.  This  has  not  been  proved  practicable.  It  is  prob- 
able that  the  bacilli  already  present  in  the  meat  would  in  some  cases 
fix  all  the  agglutinins  present,  if  stored  for  any  length  of  time. 
Practicable  methods  of  prophylactic  or  curative  immunization 
have  not  been  demonstrated. 

Bacteriologic  Diagnosis. — The  organism  may  be  demonstrated 
by  plate  cultures  from  infected  flesh.  In  man  the  disease  may  be 
diagnosed  by  the  agglutination  test,  although  with  difficulty,  for, 


INTESTINAL   OR  COLON-TYPHOID   GROUP  271 

as  has  been  noted  above,  the  various  strains  agglutinate  differently, 
and  blood  from  a  typhoid  or  a  paratyphoid  patient  may  show  a 
marked  capacity  to  agglutinate  B.  enteritidis. 

Transmission  and  Prophylaxis. — Probably  a  large  proportion 
of  the  cases  of  so-called  ptomain-poisoning  is  due  to  infection  with 
Bacillus  enteritidis  and  to  its  toxic  products  of  metabolism.  Such 
infection  undoubtedly  occurs  frequently  enough  to  justify  rigor- 
ous measures  for  its  prevention.  Meat  or  milk  from  animals 
showing  severe  gastro-intestinal  disturbances  should  never  be 
used,  as  the  infection  in  the  human  has  in  several  well-authenti- 
cated instances  been  traced  directly  to  such  practices.  Probably 
most  cases  of  meat-poisoning  originate  from  use  of  flesh  of  diseased 
animals,  but  the  possibility  of  infection  with  the  organism  after  the 
animal  has  been  slaughtered  should  not  be  ignored.  Experiments 
have  shown  that  when  fresh  meat  is  inoculated  upon  the  surface 
with  a  culture  of  B.  enteritidis,  the  organism  rapidly  penetrates 
the  tissues,  even  at  low  temperatures.  Such  infection  might 
easily  occur  in  unsanitary  abattoirs,  through  flies  and  careless 
handling. 

It  should  be  noted  that  certain  types  of  the  paratyphoid 
bacillus  are  very  similar  to  this  form,  if  not  identical  with  it,  and 
doubtless  are  the  cause  of  meat-poisoning  as  well. 

Bacillus   choleras  suis 

Synonyms. — Bacillus  suipestifer;  B.  salmoni;  Salmonella. 

Salmon  and  Smith,  in  1885,  described  what  is  known  as  the 
Bacillus  cholera  suis  as  the  cause  of  the  disease  called  by  them 
swine  plague.  In  the  following  year  Smith  discovered  another 
organism  associated  with  a  different  disease  of  swine.  This  led  to 
a  revision  of  terminology,  wrhich  has  since  come  into  common  use, 
and  the  organism  first  described  is  now  known  as  the  hog-cholera 
bacillus.  Smith  recovered  this  organism  from  the  spleens  of 
about  500  hogs  affected  with  hog-cholera.  It  was  quite  generally 
accepted  as  the  cause  of  the  disease,  until  de  Schweinitz  and  Dorset 
reported  an  outbreak  of  hog-cholera  in  which  the  B.  cholera  suis 
was  not  the  primary  cause.  This  was  shown  by  the  transmission 
of  the  disease  by  blood  filtered  through  fine-grained  porcelain 
bougies,  a  procedure  which  removed  the  bacillus  completely,  as 


272 


VETERINARY   BACTERIOLOGY 


shown  by  the  fact  that  the  filtrate  was  quite  incapable  of  infecting 
culture-media.  By  the  subsequent  work  of  Dorset,  Bolton,  and 
McBryde  it  was  shown  quite  conclusively  that  the  B.  cholerce  suis 
is  not  the  cause  of  hog-cholera  in  the  Mississippi  Valley,  and  but  a 
secondary  invader  at  most.  Hog-cholera  and  its  virus  will  be 
considered,  therefore,  under  the  heading  of  Diseases  Caused  by 
Ultramicroscopic  Organisms.  The  Bacillus  cholerce  suis,  however, 
doubtless  plays  some  part  in  the  disease  as  a  secondary  invader, 
and  is,  therefore,  worthy  of  consideration. 

Morphology    and    Staining. — This   organism    differs   morpho- 
logically in  no  essential  character  from  B.  enteritidis. 

Isolation  and  Culture. 
— B.  choleroe  suis  may 
frequently  be  isolated  at 
once  in  pure  culture  from 
the  organs  of  infected 
animals,  particularly 
from  the  spleen.  It  has 
likewise  been  isolated 
by  plating  the  intestinal 
contents  of  normal  and 
infected  animals.  The 
organism  grows  upon 
agar  and  gelatin,  form- 
ing a  grayish,  glistening, 
non-viscid  growth,  which 
is  not  particularly  char- 
acteristic. No  growth 
occurs  upon  potatoes 

having  a  decided  acid  reaction,  but  upon  those  which  are  neutral 
or  alkaline  a  thin,  glistening,  usually  yellowish,  layer  is  formed. 
Bouillon  is  uniformly  clouded;  a  slight  pellicle  may  appear  in 
time.  A  grayish,  friable  sediment  is  formed.  Milk  shows  a 
slight  initial  acidity,  but  soon  becomes  alkaline,  and  gradually 
becomes  opalescent  and  finally  translucent.  It  will  be  noted  that 
there  are  no  marked  cultural  differences  between  B.  enteritidis 
and  B.  choleras  suis. 

Physiology.— B.  choleroe  suis  is  aerobic  and  facultative  anaerobic. 


Fig.  112. — Bacillus  cholerce  suis,  organisms 
from  a  young  culture  (deSchweinitz,  Bureau 
Animal  Industry). 


INTESTINAL  OR   COLON-TYPHOID   GROUP 


273 


The  optimum  growth  temperature  is  about  37°;  it  grows  also, 
but  more  slowly,  at  room-temperature.  The  thermal  death- 
point  is  58°,  with  ten  minutes  exposure.  The  organism  will 
remain  viable  for  several  days  when  dried.  Gas  and  acid  are 


produced  in  dextrose  broth.     The  gas  formula  is 


TT 


reverse  of  that  of  B.  coll.  Lactose  and  saccharose  are  not  fer- 
mented, and  no  growth  occurs  in  the  closed  arm  of  the  fermentation 
tube  containing  these  sugars.  Indol  is  not  ordinarily  produced. 


Fig.  113. — Bacillus  cholera  suis,  showing  flagella  (deSchweinitz,  Bureau  Animal 

Industry). 

It  will  be  noted  that  there  are  no  physiologic  differences  between 
the  B.  enteritidis  and  B.  choleroe  suis. 

Pathogenesis. — It  should  again  be  emphasized  that  the  B. 
chokrce  suis  is  not  the  primary  cause  of  hog-cholera,  but  that  it 
is  a  secondary  invader  of  importance,  and  may  be  occasionally 
the  primary  cause  of  disease  in  hogs,  but  this  disease  probably 
would  possess,  according  to  Dorset,  Bolton,  and  McBryde,  a  low 
degree  of  contagiousness. 

Experimental  Evidence  of  Pathogenesis. — Some  differences  in 
virulence  have  been  observed  in  cultures  obtained  from  different 
sources.  Rabbits  succumb  to  septicemia  in  five  to  eight  days 

18 


274  VETERINARY   BACTERIOLOGY 

when  inoculated  with  yV  c.c.  of  a  virulent  bouillon  culture.  Guinea- 
pigs  are  more  refractory,  and  die  after  seven  to  twelve  days. 
Subcutaneous  and  intravenous  injections  and  feeding  experi- 
ments rarely  produce  death  in  the  hog.  The  animal  may  some- 
times show  fever  and  depression,  particularly  after  the  intravenous 
inoculation,  but  the  infection  is  rarely  fatal  unless  1  or  2  c.c.  or 
more  of  culture  are  used.  Feeding  with  large  quantities  of  culture 
or  long-continued  feeding  sometimes  proves  fatal. 

Character  of  Disease  and  Lesions  Produced. — An  examination 
of  a  rabbit  killed  by  injections  of  B.  cholerce  suis  shows  lesions 
differing  in  no  material  respect  from  those  discussed  under  B.  en- 
teritidis.  To  just  what  extent  the  characteristic  lesions  in  hog- 
cholera,  particularly  in  the  chronic  types,  are  due  to  infection 
by  this  organism  is  uncertain.  It  is  probable  that  in  many  cases, 
at  least,  it  is  responsible  for  the  development  of  intestinal  ulcers. 
Inasmuch  as  it  is  sometimes  found  in  the  blood  of  animals  infected 
by  hog-cholera,  it  is  probable  that  death  may  be  due  directly  to 
their  activity. 

Immunity. — The  topic  of  immunity  against  hog-cholera  will  be 
considered  under  that  heading.  The  B.  cholerce  suis  produces  no 
true  toxin,  but  there  is  some  evidence  of  the  formation  of  endo- 
toxin.  Agglutinins  are  present  normally  in  the  blood  of  the  hog, 
and  immune  agglutinins  may  be  produced  by  the  systematic  im- 
munization of  animals  by  killed  cultures.  Group  agglutination 
with  other  members  of  this  subgroup  has  been  demonstrated.  It 
has  also  been  shown  that  immunization  of  the  hog  against  true 
hog-cholera  results  in  a  considerable  increase  of  agglutinins  for  B. 
cholerce  suis  in  the  blood.  Opsonins  for  B.  cholerce  suis  have  been 
shown  to  be'present  in  normal  serum.  Since  the  discovery  of  the 
filterable  virus,  efforts  at  immunization  by  the  use  of  vaccines  and 
sera  prepared  by  the  use  of  B.  cholerce  suis  have  been  practically 
abandoned. 

Bacillus   paratyphosus 

Synonym. — Paratyphoid  or  paracolon  bacillus. 

Disease  Produced. — Paratyphoid  in  man,  possibly  similar 
infections  in  animals. 

Gwyn,  in  1898,  isolated  from  a  clinical  typhoid  case  an  organ- 
ism which  belonged  to  the  intermediate  subgroup  of  intestinal 


INTESTINAL   OR   COLON-TYPHOID   GROUP  275 

organisms,  rather  than  to  the  typhoid-dysentery  group.  Similar 
organisms  have  been  isolated  repeatedly  since  that  time — in  some 
instances  from  typical  typhoid  cases,  in  others  from  cases  that  had 
all  the  clinical  symptoms  of  typhoid,  but  that  did  not  give  the 
agglutination  reaction. 

Morphology  and  Staining. — This  organism  corresponds  closely 
in  morphologic  and  staining  characters  to  the  Bacillus  enteritidis 
and  the  Bacillus  cholera  suis. 

Isolation  and  Culture. — The  organism  has  been  isolated  in 
pure  culture  directly,  from  the  blood,  and  by  plate  cultures 
from  the  internal  organs  in  disease,  and  particularly  from  the 
intestinal  contents  of  man  and  of  the  lower  animals.  In  gen- 
eral cultural  characters  the  organism  resembles  the  B.  enteri- 
tidis. It  has  been  found  in  practice  that  two  varieties  may  be 
differentiated,  termed  A  and  B,  respectively.  Type  A  does  not 
produce  a  terminal  alkalinity  in  milk  and  dissolve  the  casein, 
and  in  that  respect  differs  from  B.  enteritidis,  while  type  B  corre- 
sponds exactly. 

Physiology. — Not  markedly  different  from  B.  cholera  suis. 

Pathogenesis. — Paratyphoid  fever  in  man  has  been  attended 
by  a  low  mortality;  in  consequence  few  autopsies  have  been  re- 
ported. In  both  animals  and  man  infection  partakes  more  of  the 
nature  of  an  acute  enteritis  than  does  typhoid  fever;  the  lymph- 
atics are  not  generally  invaded  as  in  typhoid,  and  the  Peyer's 
patches  are  not  swollen  and  ulcerated. 

Immunity. — Probably  an  endotoxin,  less  soluble,  but  in  some 
respects  similar  to  that  of  B.  enteritidis,  is  produced  by  these 
organisms.  Agglutinins  are  produced  in  infected  individuals. 
The  agglutination  reactions  of  types  A  and  B  differ  markedly. 
It  was  this  difference  which  first  suggested  the  existence  of  the  two 
types.  No  method  of  practical  immunization  against  the  disease 
is  known. 

Bacteriologic  Diagnosis. — The  differentiation  of  paratyphoid 
may  be  made  clinically  by  the  specific  agglutination  tests.  The 
absence  of  a  test  in  a  case  of  clinical  typhoid  calls  for  a  repetition 
with  the  two  types  of  paratyphoid  bacilli. 

Transmission. — It  is  probable  that  certain  gastro-intestinal 
infections  in  cattle  may  be  caused  by  organisms  of  this  type,  and 


276 


VETERINARY   BACTERIOLOGY 


that  meat  and  milk  may  become  contaminated  from  these  sources. 
Milk  and  meat  are  probably  the  most  common  sources  of  infection, 
although  water  has  been  clearly  shown,  in  some  instances,  to  be  the 

source  of  epidemics. 

Bacillus   psittacosis 

Nocard,  in  1892,  isolated  an  organism  belonging  to  the  inter- 
mediate group  from  cases  of  psittacosis  (Latin,  psittacus,  parrot), 
a  type  of  pneumonia  supposed  to  be  contracted  from  infected 
parrots.  The  same  organism  has  since  that  time  been  isolated 
from  other  outbreaks.  In  cultural,  morphologic,  and  physiologic 
characters  it  is  scarcely  to  be  differentiated  from  the  B.  enteritidis. 
Some  differences  have  been  found  in  agglutinating  properties  of 
the  specific  sera,  and  this  organism  is  believed,  on  these  grounds, 
by  some  authors  to  constitute  a  distinct  species.  The  disease  is 
uncommon,  and  is  of  little  importance. 

Bacillus  typhi  murium 

Loftier,  in  1889,  described  an  organism  as  the  cause  of  an  epi- 
demic among  the  mice  kept  for  experimental  purposes.  Danysz, 

in  1900,  described  a 
similar,  probably  iden- 
tical, organism,  and 
recommended  its  use  in 
the  destruction  of  rats. 
These  forms  have  all  the 
morphologic  and  cultural 
characteristics  of  the 
enteritidis  group,  but 
show  some  differences  in 
pathogenicity  and  in 
formation  of  specific 
agglutinins.  Cultures  of 
these  organisms  have 
Fig.  114.— Bacillus  typhi  murium  (Migula).  been  widely  exploited  as 

specific     for    mice    and 

rats,  producing  a  rapidly  fatal  disease,  but  as  harmless  to  the 
Higher  animals.  Reports  as  to  their  efficacy  when  fed  to  the 
vermin  are  conflicting;  the  results  seem  in  some  cases  to  have 


INTESTINAL   OR   COLON-TYPHOID   GROUP  277 

been  favorable.  It  evidently  is  true  that  the  virulence  of  the 
organism  is  subject  to  considerable  variations,  for  the  careful 
work  of  Rosenau  showed  the  culture  which  he  possessed  to  be 
worthless  in  the  extermination  of  rats.  On  the  other  hand,  there 
is  some  evidence  that  the  organism  is  not  so  free  from  harmful 
effect  on  the  human  as  has  been  supposed.  Fatal  infections  in 
man  have  been  reported  from  Japan. 

Bacillus  pulloram 

Disease  Produced. — White  diarrhea  of  chicks. 

Rettger  and  Harvey  have  described  the  Bacillus  pullorum  as 
the  specific  cause  of  a  white  diarrhea  in  young  chicks.  Its  etio- 
logic  relation  to  the  disease  has  been  called  seriously  into  question 
by  Morse,  Hadley,  and  others,  who  believe  that  it  is  either  a  com- 
mensal or  a  secondary  invader,  and  that  the  disease  is  in  reality 
a  coccidiosis.  The  evidence  is  somewhat  conflicting  on  this  point. 
The  importance  of  the  B.  pullorum,  even  as  a  secondary  invader, 
renders  a  description  pertinent. 

Morphology  and  Staining. — Bacillus  pullorum  is  a  rod,  0.3  to 
0.5  by  1  to  2.5  ft,  with  rounded  ends.  It  occurs  singly  or  very 
rarely  in  chains.  It  is  non-motile,  does  not  produce  capsules  or 
spores.  It  stains  readily  and  uniformly  with  ordinary  aqueous 
anilin  dyes,  and  is  gram-negative. 

Isolation  and  Culture. — The  organism  may  be  isolated  from  the 
infected  chicks  by  opening  the  body  with  aseptic  precautions, 
and  making  streaks  upon  the  surface  of  agar  slants  with  blood 
or  the  pulp  of  the  spleen  or  liver.  Upon  the  agar  slant  the  colonies 
are  discrete,  and  at  first  resemble  the  pin-point,  translucent 
colonies  of  the  Streptococcus.  They  enlarge  later.  Upon  gelatin 
the  colonies  resemble  those  of  the  typhoid  bacillus.  Little  growth 
occurs  upon  potato.  Milk  is  a  suitable  medium,  but  there  is 
little  change,  no  coagulation,  and  no  proteolysis. 

Physiology. — The  organism  is  aerobic  and  facultative  anaerobic. 
The  optimum  growth  temperature  is  about  37°.  Dextrose  and 
mannite  are  fermented,  with  the  production  of  both  acid  and  gas. 
Maltose,  lactose,  and  saccharose  are  not  fermented.  Indol  is 
not  produced. 

Pathogenesis. — Experimental  Evidence. — Rettger  has  isolated 


278  VETERINARY   BACTERIOLOGY 

the  specific  organism  in  several  outbreaks  of  the  disease  from  the 
internal  organs,  particularly  the  livers,  of  chicks  that  had  died 
of  the  disease  or  were  showing  symptoms.  He  also  isolated  it 
from  abnormal  egg-yolks  in  the  ovaries  of  hens,  from  freshly  laid 
eggs,  and  from  the  yolk-sacs  of  fully  developed  chicks  within  the 
shell.  He  also  succeeded  in  infecting  chicks  by  feeding,  but  the 
disease  was  not  always  contracted.  Subcutaneous  injections 
always  proved  fatal.  Hadley,  Kirkpatrick,  and  others  have  been 
unsuccessful  in  transferring  the  disease  by  feeding. 

Characteristics  of  Disease  and  Lesions. — The  most  noticeable 
antemortem  characteristics  are  emaciation  and  wasting  of  the  chick, 
and  the  white  diarrhea.  The  lesions  are  confined  principally  to 
the  intestines,  and  particularly  the  cecum.  The  liver  is  some- 
times congested  in  areas. 

Immunity. — Practicable  methods  of  immunization  have  not 
been  evolved. 

Bacteriologic  Diagnosis. — The  organism  may  be  isolated  in 
pure  culture  from  the  internal  organs,  particularly  the  liver. 
Work  on  the  normal  intestinal  flora  of  the  chicken  is  needed. 

Transmission  and  Prophylaxis. — Rettger  claims  that  the  dis- 
ease is  sometimes  present  before  hatching,  the  organism  being 
present  in  the  ovaries  and  oviduct,  and  that  contamination  of  the 
food  likewise  results  in  infection. 

SUBGROUP  m— TYPHOID-DYSENTERY  SUBGROUP 

The  three  important  organisms  belonging  to  this  subgroup — 
Bacillus  typhosus,  B.  dysenterice,  and  B.  fcecalis  alkaligenes — are 
not  ordinarily  pathogenic  for  the  lower  animals.  They  are,  how- 
ever, pathogenic  for  man,  and  since  many  of  our  diagnostic  methods 
for  other  diseases  have  been  discovered  through  their  study,  they 
are  discussed  briefly. 

Bacillus    typhosus 

Synonyms. — Bacillus  typhi;  B.  typhi  abdominalis;  Eberth  or 
Eberth-Gaffky  bacillus. 

Disease  Produced. — Typhoid  fever  in  man. 

Eberth,  in  1880,  discovered  the  B.  typhosus  in  the  spleen  and 
other  internal  organs  of  the  body  of  persons  who  had  died  of 
typhoid  fever.  Gaffky,  in  1884,  cultivated  the  organism.  It 


INTESTINAL   OR   COLON-TYPHpID   GROUP 


279 


is  now  generally  conceded  to  be  the  cause  of  typhoid  fever,  al- 
though the  experimental  animals  cannot  ordinarily  be  infected. 

Distribution. — Typhoid  fever  is  widely  distributed  throughout 
temperate  and  tropical  countries.  It  is  constantly  present,  fre- 
quently in  epidemic  form,  in  the  United  States. 

Morphology. — Bacillus  typhosus  is  a  short,  plump  rod,  usually 
varying  between  0.5  and  0.8  (J-  in  diameter,  and  1  to  3  ^  in  length. 
It  is  motile  by  means  of  numerous  flagella.  It  does  not  produce 
capsules  or  spores.  It  stains  readily  with  aqueous  anilin  dyes. 
Granular  staining  is  some- 
times observed,  although  the 
cells  usually  stain  uniformly. 
It  is  gram-negative. 

Isolation  and  Culture. — 
The  desirability  of  isolating 
B.  typhosus  from  contamin- 
ated water  has  led  to  the 
development  of  many  media 
in  an  effort  to  accomplish 
this.  A  quantitative  esti- 
mation of  the  typhoid  bacil- 
lus from  such  sources  does 

not  seem  to  be  practicable, 

Pig.  llo. — Bacillus  typhosus,  clump  in 

but    the    qualitative    deter-     a    section   of    a    spleen    (Frankel    and 
mination  of  presence  may  be     Pfeiffer). 
carried    out.     The    methods 

used  are  to  inhibit  the  growth  of  the  purely  saprophytic  organ- 
isms present  by  the  use  of  antiseptic  substances,  such  as  mala- 
chite green,  caffeine,  and  crystal  violet,  and  to  incubate  such 
media  at  blood-heat.  These  media  do  not  inhibit,  in  general, 
the  growth  of  either  B.  typhosus  or  B.  coli,  and  dependence  is 
placed  upon  differences  in  colony  characters  and  media  reactions 
to  separate  them  in  subsequent  plating. 

The  colonies  upon  gelatin  are  somewhat  smaller  and  more 
delicate  than  those  of  the  B.  coli.  This  organism  was  originally 
described  as  producing  a  thin,  "  invisible  growth  "  upon  potato. 
This  is  true  upon  potato  with  an  acid  reaction,  but  upon  alkaline 
or  neutral  potato  the  growth  is  relatively  abundant. 


280  VETERINARY   BACTERIOLOGY 

Physiology. — B.  typhosus  develops  best  at  a  temperature  of 
37°,  but  will  grow  at  room-temperature.  It  is  an  aerobe  and 
facultative  anaerobe.  No  indol  is  produced.  Acid,  but  no 
gas,  is  formed  from  dextrose.  Neither  acid  nor  gas  is  produced 
from  lactose  or  saccharose.  There  may  be  slight  initial  acidity  in 
milk,  but  there  is  never  coagulation  of  the  casein.  Proteolytic 
enzymes  are  not  developed  in  cultures. 

Pathogenesis. — Experimental  Evidence. — The  lesions  typical  of 
typhoid  in  man  are  not  produced  either  by  injection  or  feeding 
experiments  upon  the  laboratory  animals.  The  symptoms  after 
intraperitoneal  injection  of  a  guinea-pig  do  not  differ  greatly 
from  those  produced  by  the  Bacillus  coli.  Feeding  experiments 


**  ,  ~*  p  Jr    .        .          u 

m^  :^--.''-v^fl* " 

D 


•V  ^f 

•%•  »• 


Fig.  116. — Bacillus  typhosus,  showing    Fig.  117. — Bacillus  typhosus,  colony  on 
flagella  (Gunther).  agar  (Gunther). 

with  anthropoid  apes  within  recent  years  have  shown  the  possi- 
bility of  producing  the  typical  lesions  of  the  disease  in  these 
animals.  Infection  with  typhoid  bacilli  in  laboratory  workers 
has  several  times  occurred  following  the  accidental  ingestion  of 
pure  cultures  of  the  organism. 

Character  of  Lesions  and  Disease  Produced. — Clinical  diagnosis 
of  typhoid  is  frequently  difficult,  as  the  characteristics  of  the  dis- 
ease are  often  not  well  marked.  The  organism  invades  the  in- 
testinal lymph-system,  and  particularly  the  Peyer's  patches. 
The  latter  become  ulcerated,  and  perforation  of  the  intestinal  wall 
is  a  not  uncommon  result.  The  spleen  is  swollen.  The  bacteria 


INTESTINAL   OR   COLON-TYPHOID   GROUP  281 

are  usually  found  in  the  blood,  though  not  commonly  in  large  num- 
bers, but  are  abundant  in  the  spleen.  Cystitis,  cholecystitis,  and 
bone  metastases  are  not  uncommon  sequelae  to  the  infection. 

Immunity. — Xo  true  toxin  has  been  demonstrated  for  B. 
typhosus,  but  an  endotoxin  is  present.  Agglutinins  and  pre- 
cipitins  specific  for  the  organism  are  likewise  produced.  Bac- 
teriolysins  may  be  demonstrated  in  the  blood  of  animals  that  have 
been  artificially  immunized  by  injections  of  the  typhoid  bacillus. 
There  is  developed  in  the  body  of  an  individual  that  has  recovered 
from  typhoid  a  certain  degree  of  immunity,  but  this  disappears, 
so  that  it  is  not  unusual  for  a  person  to  have  several  attacks  of  the 
disease.  This  immunity  is  probably  both  bacteriolytic  and 
opsonic  in  nature. 

The  use  of  antisera  in  passive  immunization  against  typhoid 
and  in  curing  the  disease  has  not  proved  successful.  The  injec- 
tion of  such  sera  has  not  been  shown  to  have  either  an  immunizing 
or  a  curative  effect  in  man.  Active  immunization  by  the  injec- 
tion of  dead  or  living  bacteria  or  their  products  has,  on  the  con- 
trary, been  quite  successful.  Usually  the  organisms  are  scVaped 
from  the  surface  of  agar  cultures,  suspended  in  physiologic  salt 
solution,  and  killed  by  heat,  or  a  broth  culture  may  be  used. 
Such  injections  are,  of  course,  only  prophylactic. 

Bacteriologic  Diagnosis. — The  Widal  or  agglutination  test  is 
commonly  used  in  the  diagnosis  of  typhoid.  A  dilution  of  1 : 40 
and  higher  is  generally  made  to  minimize  the  effect  of  the  normal 
agglutinins  which  may  be  present  in  the  blood.  Both  microscopic 
and  macroscopic  tests  are  used;  the  former  is  the  more  delicate,  but 
the  latter  somewhat  more  reliable.  The  agglutinins  often  appear 
early  in  the  course  of  the  disease — usually  by  the  fifth  day  or  rarely 
later.  Blood  or  serum  for  making  the  test  may  be  either  liquid 
or  dried.  It  is  received  in  the  latter  condition  by  many  of  the 
state  and  municipal  bacteriological  laboratories.  The  bacteria 
may  be  cultivated  directly  from  the  blood  of  a  patient.  Fre- 
quently the  organisms  can  be  found  in  the  blood  somewhat  before 
the  serum  exhibits  a  marked  agglutinating  power.  Isolation  of 
the  organisms  directly  from  the  feces  is  sometimes  resorted  to  in 
an  effort  to  determine  the  occurrence  of  the  so-called  "  bacillus 


282 


VETERINARY   BACTERIOLOGY 


Transmission. — Typhoid  fever  is  contracted  from  contaminated 
drinking-water,  milk  and  other  foods,  and  by  contact,  the  fre- 
quency being  in  about  the  order  named.  Flies  probably  are  com- 
monly instrumental  in  carrying  the  organism  from  dejecta  of 
typhoid-fever  patients  to  food  materials.  The  term  bacillus 
carrier,  or  germ-carrier,  is  used  to  designate  an  individual  who  still 
harbors  a  pathogenic  organism  in  the  body  after  convalescence. 
Such  germ-carriers  are  particularly  dangerous,  as  they  may  give 
rise  to  an  epidemic  that  is  almost  impossible  to  trace  to  its  source. 
The  danger  of  milk  infection  is  probably  the  greatest  from  in- 
dividuals that  are  employed  in  dairies.  Several  epidemics  have 
been  traced  to  this  origin. 

Bacillus  dysenteriae 

Synonyms. — Bacillus  of  Shiga;  bacillus  of  Flexner. 
Disease  Produced. — Bacillary  dysentery. 
Shiga,  in  1898,  discovered  in  the  feces  of  patients  suffering  from 
dysentery  a  bacillus  which  he  believed  to  be  the  specific  cause  of 

the  disease.  Previous  to  this 
i£  had  been  shown  that  amebae 
may  cause  dysentery,  and  it 
was  when  examining  stools  for 
these  protozoa  that  Shiga  dis- 
covered this  organism.  In  1900 
Flexner  published  the  results  of 
work  in  Manila  and  described 
another  type  of  organism. 
Since  that  time  many  epi- 
demics have  been  studied,  and 
it  is  generally  believed  that 
the  type  described  by  Shiga  is 
the  more  common,  but  that  the 
bacillus  of  Flexner  occurs  in  a  certain  proportion  of  the  out- 
breaks, more  particularly  in  the  tropical  countries.  Other 
authors,  Hiss  in  particular,  have  differentiated  even  more 
groups. 

Morphology. — B.  dysenterice  and  B.  typhosus  are  practically 
indistinguishable  under  the  microscope  in  stained  mounts.  The 


Fig.  118. — Bacillus  dysenteries  (Kolle 
and  Wassermann). 


INTESTINAL   OR   COL*ON-TYPHOID   GROUP  283 

B.  dysenterice,  however,  is  non-motile.  Spores  and  capsules  are 
not  produced.  It  stains  uniformly  and  is  gram-negative. 

Isolation  and  Culture. — The  organism  may  be  isolat'ed  directly 
from  the  dejecta  by  plating.  The  cultural  characters  in  general 
closely  resemble  those  of  B.  typhosus.  Milk  is  rendered  perma- 
nently alkaline,  however.1 

Physiology. — The  physiologic  characters  of  B.  dysenterice 
closely  resemble  those  of  B.  typhosus.  The  ability  to  produce 
acid  in  solutions  of  various  carbohydrates  and  of  the  related 
alcohols  is  used  as  a  means  of  differentiation  of  the  varieties. 
Otho  listed  some  fifteen  different  types  by  this  means.  A  more 
conservative  and  valuable  classification  is  that  of  Hiss,  as  modified 
by  Shiga: 

Variety  ACID  PRODUCED  FROM 

B.  dysenterice.  Mannite.  Maltose.      Saccharose.       Dextrose. 

I.  Shiga,  type  I —  + 

II.  Park-Hiss  type 4-  4- 

III.  Flexner-Strong  type 4-  4-                 4- 

IV.  Harris-Wollstein  type 4-  +                  +                  + 

V.  Shiga,  type  II *  4-4-4- 

It  is  believed  that  type  I.  is  the  most  virulent.  Types  III. 
and  IV.  are  found  frequently  in  summer  diarrhea  of  infants. 

Pathogenesis. — Experimental  Evidence. — The  belief  that  the 
varieties  of  B.  dysenterice  are  the  more  important  etiologic  factors 
in  the  disease  is  based  upon  the  following  facts: 

1.  This -organism  in  some  one  of  its  varieties  has  been  shown 
to  be  present  with  great  constancy  in  the  patients'  excreta. 

2.  Injections  of  the  organisms  and  their  products  kill  laboratory 
animals,  particularly  rabbits,  although  typical  dysentery  is  not 
readily  induced  by  feeding  experiments. 

3.  The  blood-serum  of  a  patient  will,  in  general,  agglutinate 
in  high  dilution  the  strain  isolated  from  the  feces. 

4.  An  antiserum  has  been  successfully  used  in  the  prevention 
and  cure  of  the  disease. 

Character  of  Disease  and   Lesions   Produced. — The  intestine, 

particularly  the  colon,  is  inflamed  and  is  sometimes  ulcerated,  and 

may  even  show  diphtheritic  necrosis.     With  the  exception  of  this 

there  is  little  that  is  characteristic  of  the  disease.     Unlike  the 

1  Initial  acidity  followed  by  permanent  alkalinity. 


284  VETERINARY   BACTERIOLOGY 

typhoid  bacillus,  it  does  not  commonly  invade  the  blood  or  the 
internal  organs,  with  the  exception  of  the  mesenteric  glands. 
The  disease  is  rather  a  toxemia  than  a  bacteremia. 

Immunity. — A  soluble  toxin  has  been  demonstrated  for  the 
Shiga  type,  but  repeated  efforts  have  failed  to  show  that  any  such 
is  produced  by  the  Flexner  type.  This  poison  was  at  first  believed 
to  be  an  unusually  potent  endotoxin.  Conradi  first  demonstrated 
the  toxin  by  growing  the  organism  upon  agar,  then  suspending  it  in 
physiologic  salt  solution,  and  allowing  the  bacteria  to  undergo 
autolysis.  Later,  Rosenthal  and  others  showed  that  toxin  will 
be  produced  in  considerable  quantities  in  an  alkaline  bouillon, 
but  not  in  one  that  is  neutral  or  acid.  This  bouillon  is  either 
filtered  through  porcelain  filters,  or  0.5  per  cent,  phenol  is  added 
and  allowed  to  stand,  and  then  filtered  through  paper  until  clear. 
The  toxin  may  be  precipitated  by  ammonium  sulphate,  and  after 
dialysis  and  drying  of  such,  1  to  2  gm.  may  be  a  lethal  dose  for  a 
kilo  of  rabbit.  It  is  weakened  by  prolonged  heating  at  70°, 
and  destroyed  at  80°  to  100°.  The  rabbit  is  very  susceptible  to 
the  injection  of  the  toxin,  while  the  guinea-pig  is  relatively  resist- 
ant. The  effect  upon  the  rabbit  may  be  characterized  as  a  hemor- 
rhagic  necrotic  enteritis. 

Shiga  first  used  antisera  in  the  treatment  of  dysentery.  He 
regarded  its  curative  properties  as  wholly  bactericidal.  Todd, 
Koram,  Doerr,  and  others  have,  by  systematic  immunization  of  a 
horse,  secured  a  serum  that  neutralizes  the  toxin  actively.  This 
has  been  used  with  very  favorable  results  in  the  treatment  of  dys- 
entery caused  by  the  Shiga  bacillus. 

Bacteriologic  Diagnosis. — The  disease  may  be  recognized  by 
the  Widal  or  agglutination  test,  and  the  several  types  of  organisms 
differentiated  in  the  same  manner.  The  organism  may  likewise 
be  isolated  directly  from  the  stools  by  plating. 

Transmission. — Dysentery  is  spread  irt  much  the  same  manner 
as  typhoid,  and  the  same  preventive  measures  must  be  used. 

BACTERIA  OF  WATER  AND  WATER  PURIFICATION 

Diseases  of  man  and  animals,  particularly  those  of  the  alimen- 
tary tract,  are  frequently  transmitted  through  contaminated  or 
impure  water.  The  impurity,  so  called,  arises  from  the  presence 


INTESTINAL   OR  COLON-TYPHOID   GROUP  285 

of  sewage  or  surface  wash.  This  does  not  mean  that  every  water 
containing  sewage  is  necessarily  harmful,  but  that  the  presence 
of  the  sewage  is  an  indication  of  the  possible  and  probable  occa- 
sional presence  of  pathogenic  forms. 

Bacteriologic  examination  of  water  is  important,  for  several 
reasons.  Much  smaller  quantities  of  contaminating  organic  mat- 
ter may  be  determined  by  bacteriologic  than  by  chemical  means. 
Its  methods  may  be  used  in  the  determination  of  the  potability 
of  water-supplies,  in  tracing  a  typhoid  or  similar  epidemic  to  its 
source,  in  determining  the  efficiency  of  filters  for  water-supplies 
and  of  different  types  of  sewage-disposal  systems. 

Water  may  be  examined  bacteriologically,  either  quantita- 
tively or  qualitatively.  In  the  former  a  determination  of  the  total 
number  of  bacteria  present  in  the  water  is  made;  in  the  latter,  the 
tests  are  designed  to  determine  the  abundance  of  certain  specific 
bacteria.  The  qualitative  examination  may  be  again  divided 
into  determination  of  normal  sewage  bacteria,  particularly  B.  coli, 
and  of  specific  disease-producing  bacteria,  as  B.  typhosus.  The 
former  is  the  most  useful  examination  made  in  determining  the 
potability  of  a  water;  the  latter  is  rarely  used. 

Quantitative  Examination  of  Water. — In  general,  the  greater 
the  quantity  of  organic  and  decomposing  matter  present  in  water, 
the  greater  will  be  the  number  of  bacteria  present.  However, 
it  must  be  noted  that  changes  in  the  environment,  such  as  tem- 
perature, may  cause  great  variations  in  bacterial  content,  even 
though  the  original  water  be  uncontaminated.  For  example, 
water  from  a  source  quite  above  suspicion  may  have  less  than 
100  bacteria,  to  the  cubic  centimeter.  This  same  water,  carefully 
sampled  in  a  sterile  bottle  and  allowed  to  stand  at  room-tempera- 
ture, may  show  in  twenty-four  to  forty-eight  hours  hundreds  of 
thousands  to  the  cubic  centimeter.  It  is  important,  therefore, 
that  the  sample  taken  for  examination  shall  be  typical,  and  that 
it  be  examined  immediately,  to  prevent  multiplication  of  the 
bacteria  present. 

Media  Used. — Either  nutrient  gelatin  or  agar  may  be  used.  It 
should  be  prepared  according  to  the  methods  outlined  by  the 
American  Public  Health  Association.  The  gelatin  will,  in  general, 
give  a  somewhat  higher  count  than  the  agar,  but,  when  many 


286 


VETERINARY   BACTERIOLOGY 


liquefying  species  are  present,  the  count  on  the  gelatin  must  be 
made  before  all  the  slower-growing  species  have  had  a  chance  to 
develop. 

Methods. — Various  dilutions  of  the  water  to  be  tested  are 
placed  in  a  series  of  sterile  Petri  dishes,  and  the  melted  medium 
to  be  used  is  poured  in  and  thoroughly  mixed.  After  the  medium 
has  solidified,  the  plates  may  be  kept  at  room-temperature,  or, 
better,  placed  in  a  thermostat  which  maintains  a  temperature  of 
about  20°  to  22°.  The  number  of  colonies  developing  upon  a 
given  plate,  multiplied  by  the  dilution  introduced,  will  give 
approximately  the  number  present  per  cubic  centimeter  in  the 
original  sample.  The  final  count  should  be  made  only  after  the 
lapse  of  several  days  or  a  week.  Discrepancies  will  usually  be 
detected  between  the  numbers,  as  determined  from  the  plates 
containing  the  lesser  and  the  greater  dilutions.  It  is  customary 
to  use  the  plate  having  the  nearest  to  200  colonies  in  making  the 
final  estimation.  Where  more  than  this  number  of  colonies  are 
present,  it  is  probable  that  many  more  have  failed  to  develop  at 
all,  or  to  a  size  that  can  be  readily  detected,  on  account  of  the  over- 
crowding. The  numbers  of  bacteria  can,  of  course,  be  determined 
only  approximately  by  the  higher  dilution;  it  is,  therefore,  cus- 
tomary to  follow  the  mode  of  expression  suggested  by  the  committee 
on  standard  methods  of  the  American  Public  Health  Association: 

NUMBERS  OF  BACTERIA  FROM — 

1-50  shall  be  recorded  to  the  nearest  unit. 

51-100 

100-250 

251-500 

501-1000 

1001-10,000 

10,001-50,000 

50,001-100,000 

100,001-500,000 

500,001-1,000,000 

1,000,001-5,000,000 

Interpretation  of  Results. — No  standard  for  the  number  of 
bacteria  that  may  be  present  in  potable  water  can  be  set  because 
of  the  various  factors  which  may  determine  a  high  count.  Stern- 
berg,  however,  has  suggested  a  standard  that  is  in  general  ap- 


5 

10 

25 

50 

100 

500 

1000 

10,000 

50,000 

it 

100,000 

INTESTINAL   OR   COLON-TYPHOID   GROUP  287 

plicable;  water  containing  less  than  100  bacteria  per  cubic  centi- 
meter is  probably  pure;  one  containing  500  bacteria  is  suspicious, 
and  one  with  1000  bacteria  is  quite  certainly  bad.  The  number  of 
bacteria  normally  present  in  unpolluted  supplies  of  various  kinds 
differs  considerably;  for  example,  that  in  the  deep  wells  of  a  region 
from  those  of  its  lakes,  and  standards  must,  therefore,  be  established 
for  each.  Determination  of  numbers  is  probably  most  useful  in 
systematic  examination  of  the  efficiency  of  filtration  of  public 
water-supplies.  In  some  countries  these  tests  are  made  daily, 
and  the  maximum  bacterial  content  of  the  filtered  water  that  may 
be  used  has  even  been  fixed  by  law. 

When  gelatin  is  used,  a  separate  count  should  be  made  of  the 
colonies  which  develop  that  are  capable  of  liquefying  the  medium. 
Such  organisms  are  particularly  characteristic  of  the  surface  soil, 
and  usually  belong  to  the  Bacillus  subtilis  group.  The  presence  of 
such  in  large  numbers  is  an  index  to  the  extent  of  the  surface-wash, 
and  not  in  general  of  the  extent  of  sewage  pollution.  This  deter- 
mination is  frequently  of  value  in  the  examination  of  shallow  wells. 

Agar  plates  may  be  incubated  at  blood-heat.  The  typical 
water  bacteria  develop  very  slowly,  if  at  all,  at  this  temperature. 
A  count  made  in  forty-eight  hours  of  such  a  plate  is  a  fair  index 
of  the  amount  of  sewage  contamination  usually,  as  the  organisms 
from  this  source  thrive  best  at  this  temperature.  This  determina- 
tion, however,  is  largely  displaced  by  the  use  of  the  litmus-lactose- 
agar  plates,  as  discussed  under  Qualitative  Analysis. 

Qualitative  Examination  of  Water. — As  has  before  been  stated, 
water  may  be  examined  for  the  specific  pathogens  it  may  contain, 
or  for  the  presence  of  sewage  and  intestinal  bacteria,  particularly 
B.  coli. 

Isolation  of  Specific  Pathogens. — B.  typhosus  is  the  organism 
for  which  examinations  have  been  most  frequently  made.  It  has 
been  actually  isolated  from  water  in  a  few  instances  only.  Fre- 
quently only  a  very  small  percentage  of  the  colonies  which  develop 
from  direct  plating  of  typhoid  stools  are  typhoid  colonies.  The 
chances  of  direct  isolation  by  plating  sewage  or  water  from  a  sup- 
ply is,  therefore,  remote,  even  though  this  organism  be  present  in 
numbers  such  as  to  cause  an  epidemic  of  the  disease  among  the 
consumers. 


288  VETERINARY   BACTERIOLOGY 

Usually  the  search  for  the  specific  organism  in  the  water-supply 
is  not  begun  until  there  is  an  outbreak  of  the  disease,  and  the 
probabilities  are  that  the  organism  by  that  time  has  disappeared 
from  the  supply.  Many  methods  for  isolation  have  been  devised, 
but  are  not  commonly  used.  For  the  most  part,  they  are  de- 
pendent upon  enrichment  by  placing  the  suspected  water  in  broth 
containing  antiseptics  which  will  inhibit  the  growth  of  other  bac- 
teria, but  not  of  the  intestinal  forms.  This  material  is  then  plated, 
and  the  typhoid-like  colonies  are  fished  out  and  tested  one  at  a 
time,  the  crucial  test  usually  applied  being  the  ability  to  agglutin- 
ate with  high  dilution  of  typhoid  antiserum. 

The  specific  organism  of  Asiatic  cholera  may  be  more  readily 
isolated  than  that  of  typhoid.  Flasks  of  peptone  salt  solution  are 
inoculated  with  the  suspected  water,  and  incubated  at  blood-heat 
for  twenty-four  hours  or  less,  and  transfers  made  to  fresh  flasks 
from  the  surface  layer.  The  cholera  spirillum  has  a  considerable 
avidity  for  free  oxygen,  and  swarms  just  below  the  surface  in  much 
greater  numbers  than  elsewhere  in  the  medium.  Plates  made 
from  this  surface  film  should  show  the  characteristic  colonies. 

Isolation  of  B.  coli. — Advantage  may  be  taken  of  the  physiologic 
and  cultural  characteristics  of  the  B.  coli  to  isolate  it  from  water. 
In  examination  of  large  numbers  of  .samples  it  is  often  found 
useful  to  make  what  is  termed  a  preliminary  or  presumptive  test 
for  the  presence  of  the  colon  bacillus.  Fermentation  tubes  con- 
taining 1  per  cent,  dextrose  (glucose)  broth  are  inoculated  with 
varying  amounts  of  the  water  to  be  tested.  If  gas  is  not  produced 
in  any  of  the  tubes,  it  is  evident  that  B.  coli  is  not  present;  at  least 
in  any  considerable  numbers.  A  negative  result  is,  therefore, 
good  evidence  of  the  purity  of  the  water  examined.  A  positive 
test  makes  it  probable  that  the  water  contains  the  colon  bacillus, 
although  further  tests  are  necessary  to  establish  the  fact;  hence 
the  name,  presumptive  test.  The  evidence  that  B.  coli  is  present 
is  much  strengthened  if  gas  to  the  amount  of  30  per  cent,  or  more, 

TT  rt 

and  having  a  composition  of  ^7^~  =  7,  is  produced.  An  approxima- 
tion of  the  number  of  colon  bacilli  present  may  sometimes  be  made 
by  observing  the  dilutions  of  the  water  in  which  gas  is  produced. 
For  example,  if  gas  is  produced  in  dilutions  of  1 :  0,  1 :  10,  and  1 :  100, 


INTESTINAL   OR  COLON-TYPHOID   GROUP  289 

but  not  in  higher  dilutions,  it  may  be  inferred  that  there  are  be- 
tween 100  and  1000  B.  coli  present  per  cubic  centimeter  in  the 
original  sample. 

A  more  accurate  determination  of  the  number  of  B.  coli  present 
in  a  given  sample  may  be  secured  by  plating  different  dilutions  in 
agar  containing  1  per  cent,  lactose,  colored  blue  by  litmus  solution, 
and  incubating  twenty-four  to  forty-eight  hours  at  37°.  Organ- 
isms which  can  ferment  lactose  with  acid  production  are  surrounded 
by  a  red  discoloration  of  the  litmus.  Such  organisms  are  the 
B.  coli,  B.  lactis acrogenes,  and  Streptococcus.  The  first  two  may  be 
considered  together,  as  they  come  from  the  same  source  and  in- 
dicate the  same  facts.  The  colonies  of  these  may  usually  be 
readily  differentiated  from  those  of  Streptococcus  by  their  large 
size,  their  shiny  appearance,  and  the  frequent  gas-bubble  accom- 
panying the  colony  if  it  lies  below  the  surface.  The  Streptococcus 
colonies,  on  the  other  hand,  are  small,  rarely  larger  than  a  pin- 
head,  and  never  have  gas-bubbles.  It  is  sometimes  necessary 
to  make  transfers  from  colonies  and  carry  them  through  the 
various  media  to  complete  the  identification.  A  highly  contam- 
inated water  will  usually  reveal  the  acid  colonies  directly  upon 
plating,  but  in  careful  work  it  is  sometimes  necessary  to  enrich  the 
suspension.  Plates  may  be  poured  from  fermentation  tubes 
that  show  gas-production. 

B.  coli  is  not  uncommon  in  nature;  its  constant  presence  in  the 
feces  of  most  animals  makes  it  widely  distributed.  It  is  entirely 
probable  that  the  presence  of  small  numbers  of  B.  coli  in  water  may, 
therefore,  be  without  significance  from  the  standpoint  of  potability. 
It  is  generally  regarded  in  America  that  if  B.  coli  can  be  constantly 
demonstrated  in  1  c.c.  samples  of  the  water,  this  is  an  indication 
of  recent  sewage  contamination.  When  its  presence  may  be 
demonstrated  only  by  the  use  of  larger  samples  than  1  c.c.,  the 
evidence  must  be  regarded  as  inconclusive. 

Water  Purification 

Self-purification  of  Natural  Waters. — Natural  waters,  both  run- 
ning and  impounded  (as  in  lakes  and  reservoirs) ,  gradually  free  them- 
selves from  organic  and  bacterial  contamination.  The  rapidity 
and  efficiency  of  this  cleansing  process  depend  upon  many  factors. 

19 


290  VETERINARY   BACTERIOLOGY 

Sedimentation  is  probably  the  most  potent  factor  in  freeing 
a  contaminated  water  from  bacteria.  The  bacteria  themselves 
have  a  slightly  greater  specific  gravity  than  water,  and  tend  to  go 
to  the  bottom  under  the  influence  of  gravity.  This  occurs  more 
rapidly  when  other  and  larger  solid  particles  are  in  suspension; 
flocculation  and  more  rapid  sedimentation  then  frequently  occur. 
Advantage  is  taken  of  this  fact  in  the  artificial  purification  of 
water,  and  coagulants  of  different  kinds  are  added,  which  carry 
down  the  bacteria,  together  with  other  suspended  material. 
Diminution  of  food-supply  with  consequent  disappearance  of  many 
bacteria  is  likewise  important.  Probably  light  destroys  some 
organisms,  and  others  are  ingested  by  protozoa.  Some  species 
do  not  develop  in  the  presence  of  certain  other  forms,  that  is, 
they  exhibit  antibiosis.  Water-plants,  alga,  and  natural  obstruc- 
tions of  all  kinds  to  water-flow  exert  a  filtering  action.  Changes 
in  temperature  may  inhibit  the  growth  and  even  destroy  some  bac- 
teria. A  contaminated  stream  is  constantly  diluted  by  the  influx 
of  ground- water  and  of  tributaries. 

Purification  of  Drinking-water. — For  domestic  purposes,  water 
may  be  effectually  purified  by  heating  to  the  boiling-point  for  a 
few  minutes.  All  the  pathogenic  bacteria  are  quite  effectually 
eliminated  by  this  method.  Berkefeld  porcelain  filters,  if  properly 
constructed,  are  also  efficient.  They  must  be  cleaned  and  sterili- 
ized  at  short  intervals,  otherwise  the  organisms  will  grow  through 
the  pores  of  the  filter,  and  the  water  passing  through  will  be  as 
contaminated  as  the  original  supply.  Where  a  city-supply  must 
be  purified,  it  is  commonly  pumped  into  reservoirs  and  allowed 
to  settle,  with  or  without  the  addition  of  coagulants.  It  is  then 
passed  through  filters  of  various  types,  usually  sand.  Passage 
through  a  properly  constructed  filter  of  this  type  has  been  shown 
to  be  exceptionally  efficient.  Such  a  system  requires  careful 
supervision.  An  efficient  system  will  remove  over  99  per  cent, 
of  the  bacteria  originally  present.  Some  city  supplies  are  pumped 
under  pressure  through  sand-filters.  This  does  not  seem  to  be  as 
efficient  a  means  of  ridding  the  water  of  bacteria  as  the  other,  as  a 
filter,  in  any  case,  to  retain  its  highest  efficiency,  must  remain  for 
some  time  undisturbed,  and  filters  of  the  latter  type  require  fre- 
quent cleaning  and  washing.  The  installation  of  filtration  plants 


INTESTINAL   OR   COLON-TYPHOID    GROUP  291 

for  purification  of  city  supplies  has  in  many  cases  resulted  in  a 
great  diminution  of  the  death-rate  from  typhoid  and  other  intes- 
tinal diseases. 

Sewage  Disposal. — The  question  of  proper  disposal  of  sewage 
is  closely  related  to  the  topic  of  pure  water  for  domestic  purposes. 
Usually  sewage  is  allowed  to  flow  into  a  suitable  stream,  and  is 
purified  as  it  passes  down-stream.  There  is  no  valid  objection 
to  this,  providing  there  is  a  sufficient  and  constant  flow  of  water 
in  the  stream  to  insure  dilution,  and  the  water  of  this  stream  is 
not  used  as  a  city  supply  further  down.  Unfortunately,  too  little 
attention  has  been  paid  to  this  subject,  and  the  high  typhoid 
death-rates  in  some  cities  are  due  directly  to  the  use  of  such 
sewage  polluted  water.  Berlin  and  Paris  purify  their  sewage  by 
using  it  in  the  irrigation  of  large  tracts  of  land,  and  recollecting 
the  water  in  underdrains.  Such  a  system  is  highly  efficient,  but, 
as  it  requires  a  particular  type  of  soil,  large  areas,  and  suitable 
conditions,  it  is  not  often  practicable.  For  sewage  disposal  in 
small  cities  and  towns,  and  even  private  residences  or  farms,  some 
of  the  numerous  modifications  of  the  septic  tank  and  filter-bed  have 
been  shown  to  be  most  efficient.  The  sewage  is  first  carried  to  a 
septic  tank,  so  called — a  large  tank,  usually  of  brick  or  concrete, 
and  commonly  covered.  This  tank  is  planned  so  that  the  sewage 
flow  of  twelve  to  twenty-four  hours  will  fill  it,  or,  in  other  words, 
that  a  given  portion  of  sewage  will  require  that  time  to  pass 
through.  Here  much  of  the  solid  material  settles  out.  The  dis- 
solved oxygen,  if  any  be  present  in  the  raw  sewage,  is  quickly 
used  up,  and  anaerobic  conditions  are  established.  Under  such 
treatment  the  decomposition  of  the  organic  matter  occurs  rapidly. 
Most  of  the  sediment  of  the  septic  tank  is  soon  dissolved  by 
bacterial  action.  Gases,  particularly  H2S,  CH4,  H2,  and  NH3,  are 
produced.  These  rise  to  the  top,  and  are  there  intercepted  by  the 
heavy  scum  which  forms,  and  oxidized  to  H2S04,  or  free  S2,  CO2, 
H2O,  -and  HN03;  hence  there  is  but  little  disagreeable  odor  to  be 
noted  about  such  a  plant.  The  organic  material  is,  for  the  most 
part,  broken  down  into  soluble,  easily  oxidizable  substances. 
The  sewage  must  not  be  held  under  these  conditions  for  too  long  a 
period,  otherwise  the  decomposition  will  go  too  far.  The  sewage 
then  usually  passes  to  a  dosing  chamber.  This  is  simply  a  chamber 


292  VETERINARY    BACTERIOLOGY 

which  automatically  discharges  through  one  or  more  siphons  when- 
ever it  becomes  filled.  The  sewage  passes  from  here  either  into  con- 
tact beds  or  into  filter-beds.  The  former  consist  of  beds  of  crushed 
rock,  usually  with  water-tight  walls.  The  sewage  may  be  sprinkled 
over  the  surface  constantly  and  allowed  to  trickle  through  (the 
so-called  trickling  filter),  or  it  may  be  poured  onto  the  bed  in  bulk, 
and  held  in  contact  with  the  crushed  stone  for  a  time  and  then  dis- 
charged. In  either  case  the  sewage  becomes  thoroughly  aerated, 
and  the  aerobic  bacteria  rapidly  oxidize  the  organic  matter  pres- 
ent. A  filter-bed,  on  the  other  hand,  is  constructed  of  sand  under- 
laid with  gravel  and  stone.  The  sewage  is  spread  out  over  the 
surface  and  is  allowed  to  seep  through.  Opportunity  for  thorough 
aeration  of  the  sand  and  gravel  is  given  by  the  time  elapsing  be- 
tween the  discharges  from  the  dosing  chamber.  Frequently 
several  beds  are  used,  and  the  sewage  is  discharged  first  upon  one 
then  upon  another.  The  organic  material  is  retained,  probably 
by  absorption,  and,  as  the  sewage  passes  through,  the  bacteria  are 
largely  filtered  out,  and  the  organic  material  is,  for  the  most  part, 
quite  completely  oxidized.  The  water  leaving  the  drains  under 
these  filter-beds  is  relatively  pure,  in  some  cases  quite  as  pure  as 
water  from  the  average  shallow  well.  As  has  been  stated,  there 
are  many  modifications  of  the  type  of  disposal  plant.  It  has 
been  adapted  to  use  for  the  farm-house  as  well  as  the  city. 


CHAPTER  XXIX 

HEMORRHAGIC  SEPTICEMIA  GROUP 

HUEPPE,  in  1886,  united  a  number  of  organisms  causing  some- 
what similar  diseases  in  different  species  of  animals  under  the  name 
of  Bacillus  septicemice  hcemorrhagicce.  He  included  chicken 
cholera,  septicemia  of  rabbits,  swine  plague,  hemorrhagic  septi- 
cemia  of  cattle  and  of  various  wild  animals,  and  a  number  of  other 
diseases.  Trevisan  grouped  these  organisms  in  a  new  genus,  which 
he  named  Pasteurella.  The  diseases  produced  by  such  organisms 
are,  therefore,  sometimes  termed  pasteurelloses  (sing.,  pasteureU- 
osis).  Lignieres,  in  1901,  classified  the  pasteurelloses,  or  hemor- 
rhagic septicemias,  and  his  classification  has  been  generally  adopted 
by  the  French  bacteriologists,  and  is  the  outline  followed  by 
Nocard  and  Leclainche  in  their  "  Maladies  microbienne  des 
Animaux."  The  name  Bacillus  pleurisepticus  is  sometimes  used 
to  designate  the  organisms  of  this  group  as  a  whole. 

There  is  probably  no  group  of  organisms  which  is  in  greater 
need  of  thorough  study  and  revision  than  this.  Non-pathogenic 
organisms  having  the  morphologic  and  cultural  characters  of  this 
group  have  been  isolated  from  many  sources.  At  least  one  disease, 
dog  distemper,  originally  described  as  caused  by  a  Pasteurella, 
has  been  shown  to  be  due  to  an  ultramicroscopic  virus.  It  is 
possible  that  in  some  other  diseases  placed  in  this  group  the  organ- 
ism described  as  the  etiologic  factor  is  but  a  secondary  invader. 
The  discovery  that  hog-cholera  is  not  caused  by  B.  cholera  suis 
makes  it  appear  highly  probable  that  still  other  of  these  diseases 
may  be  found  to  be  due  to  a  filterable  virus.  Furthermore,  there 
has  not  been  sufficient  care  used  in  the  differentiation  of  the  various 
members  of  the  group.  It  is  not  to  be  concluded  that  our  knowl- 
edge of  none  of  these  diseases  rests  upon  a  secure  foundation; 
there  is  little  doubt,  for  example,  that  the  Bacillus  pestis  is  the 
cause  of  bubonic  plague. 

293 


294  VETERINARY   BACTERIOLOGY 

The  bacteria  belonging  to  this  group  are  closely  related  in  their 
morphologic,  physiologic,  and  cultural  characters.  A  descrip- 
tion for  one  will  answer  in  general  for  the  remaining.  They  are 
small,  plump,  short  bacilli,  with  rounded  ends,  usually  about 
0.5  to  0.8  by  1  to  2  //,  non-motile,  and  without  spores.  Capsules 
may  be  demonstrated  in  the  blood  with  some  species.  The  organ- 
isms stain  readily  with  aqueous  anilin  dyes,  but  are  gram-negative. 
The  outstanding  morphologic  character  is  the  tendency  toward 
bipolar  staining,  particularly  in  tissues.  The  ends  stain  deeply, 
the  central  portion  not  at  all,  giving  the  appearance  of  diplococci. 
The  organisms  grow  readily  upon  the  common  cultural  media, 
with  the  exception  of  potato,  upon  which  they  usually  develop 
scantily,  if  at  all,  at  blood-heat.  Gelatin  is  not  liquefied.  Some 
acid  is  produced  from  dextrose;  milk  is  usually  not  coagulated. 
The  organisms  are  not  particularly  resistant,  and  are  easily  des- 
troyed by  heat,  desiccation,  and  sunlight.  Not  only  the  organ- 
isms, but  the  diseases  produced  by  them  in  various  animals, 
resemble  each  other.  All  the  species  of  the  group  are  pathogenic 
for  the  rabbit,  and  usually  for  other  laboratory  animals.  On  the 
other  hand,  some  of  them  possess  a  high  degree  of  specificity, 
such  that  only  that  form  isolated  from  the  lesions  of  a  certain 
species  is  pathogenic  for  that  species.  The  disease  is,  in  most 
cases,  a  true  septicemia,  characterized  by  hemorrhages  in  various 
organs.  In  some  cases  it  may  be  localized  in  the  lungs,  the  lymph- 
glands,  or  the  intestines. 

As  has  been  stated,  it  is  at  present  not  possible  to  differentiate 
the  organisms  associated  with  the  various  diseases  from  each  other 
by  any  of  their  biologic  characters.  It  is,  therefore,  necessary 
to  adopt  tentatively  a  pathologic  classification,  and  group  them 
with  reference  to  the  animals  naturally  infected  and  the  diseases 
produced.  The  following  list  includes  only  the/  more  important 
types  that  have  been  described,  and  is  not  complete: 

A.  Hemorrhagic  septicemias  of  lower  animals  only. 

1.  Of  birds,  Bacillus  avisepticus. 

2.  Of  swine,  Bacillus  suisepticus. 

3.  Of  cattle,  Bacillus  bovisepticus. 

4.  Of  equines,  Bacillus  equisepticus* 

5.  Of  rabbits,  Bacillus  cuniculicida. 


HEMORRHAGIC   SEPTICEMIA    GROUP  295 

B.  Hemorrhagic  septicemia  of  rodents  transmissible  to  man: 
Bacillus  pestis. 

Bacillus  avisepticus 

Synonyms. — Bacillus  cholera  gallinarum;  B.  choleras;  Bacterium 
avicidum. 

Disease  Produced. — Fowl  or  chicken  cholera  in  domestic  fowls 
and  other  birds. 

Perroncito,  in  1878,  first  observed  this  organism  in  an  outbreak 
of  chicken  cholera.  Pasteur,  in  1880,  cultivated  the  organism 
and  studied  it  quite  at  length.  It  was  with  this  organism  that  he 
performed  his  first  experiments  upon  vaccination  and  the  prepara- 
tion of  attenuated  cultures.  It  is,  therefore,  of  considerable 
historic  interest,  as  marking  the 
beginning  of  the  experimental 
study  of  immunity. 

Distribution. — The  disease  is 
known  to  occur  in  various  Euro- 
pean countries,  and  has  been 
reported  from  Canada  and  the 
United  States. 

Morphology  and  Staining. 
— B.  avisepticus  is  a  typical 
member  of  the  hemorrhagic 
septicemia  group;  the  char- 
acters given  under  the  intro- 

Fig.  119. — Bacillus  avisepticus,  from 

ductory  heading  will  serve  for       an  agar  slant  (X  1000)  (Guntherj. 
this  form. 

Isolation  and  Culture. — The  organism  may  be  isolated  in  pure 
culture  from  the  blood  and  internal  organs  of  infected  birds.  The 
colonies  upon  gelatin  plates  are  small,  white,  usually  irregular 
dots,  without  marked  or  distinctive  characters.  In  gelatin  stab, 
growth  occurs  along  the  line  of  inoculation  in  the  form  of  numerous 
tiny,  discrete  colonies,  and  upon  the  surface  a  thick  mass  generally 
forms,  rather  circumscribed,  giving  to  the  whole  the  appearance 
of  a  nail.  Agar  and  blood-serum  slants  show  a  moderately  luxur- 
iant, white,  glistening  growth.  Bouillon  is  slightly  clouded. 

Physiologic  Characters. — B.  avisepticus  is  aerobic  and  faculta- 
tive anaerobic.  It  grows  best  at  blood-heat,  but  will  develop 


296  VETERINARY   BACTERIOLOGY 

readily  in  culture-media  at  room-temperatures.  Acid  is  produced 
from  saccharose  and  dextrose,  but  not  from  lactose;  gas  is  never 
formed.  No  proteolytic  enzymes  are  produced.  Indol  and 
phenol  are  formed  in  Dunham's  solution. 

Pathogenesis. — Experimental  Evidence. — The  B.  avisepticus 
is  pathogenic  for  chickens,  geese,  pigeons,  and  other  birds,  mice, 
and  rabbits,  producing  a  rapidly  fatal  septicemia  when  intro- 
duced subcutaneously.  When  fed,  it  will  also  produce  disease  in 
these  animals.  Guinea-pigs  appear  to  be  relatively  immune, 


p 
Fig.  120. — Bacillus  avisepticus,  in  pigeon's  blood  (Frankel  and  Pfeiffer). 

except  when  injected  with  very  large  doses.     It  is  not  known  to 
produce  disease  in  man. 

Character  of  Disease  and  Lesions  Produced. — When  introduced 
subcutaneously,  there  is  an  extensive  edema  at  the  site  of  inocula- 
tion, generally  accompanied  by  more  or  less  hemorrhage.  The 
spleen  and  liver  are  swollen  and  congested,  the  lungs  have  congested 
areas,  and  the  intestines  show  an  inflamed  mucosa,  with  occa- 
sional ulcer  formation.  Minute  hemorrhages  are  found  in  various 
organs,  particularly  the  lungs,  the  walls  of  the  intestines,  and  the 
heart.  Feeding  experiments  result  in  a  localization  of  the  lesions  in 
the  intestines.  Areas  of  necrosis  are  frequently  found  in  the  liver. 


HEMORRHAGIC   SEPTICEMIA   GROUP  297 

Immunity. — No  true  toxin  has  been  demonstrated  for  the 
B.  avisepticus.  Endotoxins  are  produced.  One  attack  of  the 
disease  with  recovery  confers  immunity.  Agglutination  in  dilu- 
tions of  1 :  6000  has  been  shown  with  blood  of  animals  artificially 
immunized.  The  nature  of  this  immunity  is  not  certainly  known, 
although  opsonins  have  been  demonstrated. 

Pasteur,  in  1880,  worked  out  a  method  of  prophylaxis  by  the 
use  of  vaccines  prepared  from  attenuated  cultures.  He  attenu- 
ated the  organism  by  long-continued  cultivation  upon  artificial 
media.  Broth  cultures  were  allowed  to  stand  from  three  to  ten 
months.  Under  these  conditions  the  virulence  is  gradually  lost, 
and  inoculation  into  the  fowl  is  followed  by  a  mild  local  reaction 
only.  This  immunizes  against  subsequent  injections  of  the 
virulent  form.  Pasteur  believed  that  the  attenuating  factor  was 
the  abundant  presence  of  oxygen,  for  cultures  which  he  sealed 
from  the  free  entrance  of  air  he  found  to  retain  their  virulence 
even  after  ten  months.  He  also  found  that  various  strains  showed 
great  differences  in  their  rate  of  attenuation.  The  Pasteur  method 
of  vaccination  has  never  come  into  general  use.  Tests  have  shown 
that  the  use  of  the  vaccine  sent  out  by  the  Pasteur  Institute  was 
apt  to  produce  typical  cholera  in  some  fowls.  It  has  been  shown 
that  some  degree  of  immunity  is  conferred  by  the  injection  of 
killed  cultures  of  the  organism. 

It  has  also  been  found  that  immunization  against  one  of  the 
members  of  the  hemorrhagic  septicemia  group  immunizes  like- 
wise against  others.  Injections  of  the  Bacillus  bovisepticus,  for 
instance,  will  protect  against  subsequent  injection  with  Bacillus 
avisepticus.  Ligniere  has  prepared  a  polyvalent  vaccine  by  grow- 
ing at  42°  to  43°  organisms  isolated  from  sheep,  cattle,  horses, 
dogs,  hogs,  and  fowls  in  bouillon.  When  allowed  to  grow  for  five 
days,  it  constitutes  the  Vaccine  I.;  for  two  days  only,  Vaccine  II. 
One-eighth  c.c.  of  I.  is  injected,  and  twelve  to  fifteen  days  later 
the  same  amount*  of  Vaccine  II.  By  this  means  he  claims  to  be 
able  to  immunize  against  all  types  of  hemorrhagic  septicemia  in 
animals  other  than  fowls.  This  method  has  not  been  utilized  in 
practice,  although  a  few  recorded  tests  have  been  favorable.  In 
summary,  therefore,  it  may  be  stated  that  no  practicable  method 
of  vaccination  against  fowl  cholera  has  been  evolved. 


298  VETERINARY   BACTERIOLOGY 

Kitt  and  Mayr,  in  1897,  showed  that  it  is  possible  to  secure  a 
protective  serum  from  the  horse  and  other  animals,  as  goat  and 
swine,  by  injections  of  living  fowl-cholera  organisms,  and  this 
serum,  when  injected  in  suitable  quantities  into  susceptible  ani- 
mals, will  protect  them  from  injections  of  virulent  bacilli.  Schrei- 
ber,  in  1899,  elaborated  upon  an  observation  of  the  preceding  in- 
vestigation that  animals  immunized  against  swine-plague  bacilli 
(B.  suisepticus)  were  likewise  immune  to  fowl  cholera.  In  1902 
he  gave  the  name  "  septicidin  "  to  a  polyvalent  serum  which  he 
prepared  by  immunization  with  B.  suisepticus,  B.  avisepticus,  and 
B.  cholerce  suis.  The  reports  relative  to  the  efficiency  of  this 
serum  are  conflicting.  It  has  not  come  into  general  use.  Hertel, 
in  1902,  reported  that  by  intravenous  injections  of  dead  bacteria 
followed  by  living  bacteria  into  an  ass  he  secured  a  serum  which  in 
injections  of  0.5  c.c.  protected  pigeons  against  10,000  times  the 
normal  lethal  dose  of  the  organism.  Ligniere  and  Spitz  have  also 
prepared  a  polyvalent  serum,  using  the  Ligniere  vaccine  noted 
above.  There  is  no  record  of  a  practical  utilization  of  this  method. 
Kitt  and  others,  in  1904,  have  described  sera  which  protect  fowls 
experimentally  inoculated  with  B.  avisepticus,  but,  like  the  others 
described,  these  have  not  come  into  general  use.  It  may  be  con- 
cluded, therefore,  that  while  immunization  against  fowl  cholera, 
either  by  vaccination  or  the  use  of  antisera,  has  been  shown  to  be 
possible,  it  has  not  been  proved  practicable. 

Bacteriologic  Diagnosis. — Stained  mounts  of  the  blood  which 
reveal  the  presence  of  gram-negative  bacilli  showing  prominent 
bipolar  staining  are  diagnostic.  The  bacillus  may  be  readily  iso- 
lated in  artificial  media.  Whether  or  not  serum  reactions,  par- 
ticularly agglutination,  might  be  utilized  in  diagnosis  is  not  known. 

Transmission. — The  disease  is  supposed  to  be  transmitted  from 
bird  to  bird  by  ingestion  of  food  or  water  fouled  with  excretions 
containing  the  specific  organism. 

Bacillus  suisepticus 

Synonym. — Bacillus  suicida. 

Disease  Produced. — Swine  plague;  Schweineseuche. 
Loffler  and  Schiitz,  in  1886,  published  results  which  established 
the  identity  of  swine  plague  as  a  specific  disease  by  the  discovery 


HEMORRHAGIC    SEPTICEMIA    GROUP 


299 


of  the  causal  organism.  In  the  same  year  Smith  isolated  what 
proved  to  be  the  same  organism  from  hogs  in  the  United  States. 
Since  that  time  it  has  been  isolated  from  animals  in  many  parts  of 
Europe  and  the  United  States.  From  the  beginning  the  close 
relationship  between  the  swine-plague  and  fowl-cholera  bacilli 
was  recognized.  In  the  early  literature  of  swine  diseases  in 
America  there  is  much  confusion  relative  to  the  use  of  the  terms 
swine  plague  and  hog-cholera.  The  discovery  that  hog-cholera  is 
caused  primarily  by  an  ultramicroscopic  organism  has  made  neces- 
sary a  very  careful  retraversing  of  the  knowledge  relative  to  swine 
plague,  and  it  has  been 
urged  that  probably  the 
B.  suisepticus  is  a  second- 
ary invader  merely,  as  is 
the  hog-cholera  bacillus, 
and  that  the  two  diseases 
differ  not  at  all  in  their 
primary  cause.  The  evi- 
dence at  present  seems  to 
point,  however,  to  a  spe- 
cific disease  caused  by  B. 
suisepticus,  and  entirely 
distinct  from  hog-cholera. 
The  question  cannot  be 
said  to  be  satisfactorily 
settled  at  the  present 

time.  In  the  United  States  it  seems  probable  that  swine  plague, 
if  such  really  exists,  is  relatively  unimportant  in  comparison  with 
hog-cholera. 

Distribution. — Swine  plague  is  known  to  occur  in  Germany 
and  in  parts  of  the  United  States.  It  has  been  reported  from 
practically  all  the  States,  but  the  evidence  is  in  most  cases  quite 
inconclusive,  as  the  final  criterion  must  be  the  isolation  of  the 
specific  organism. 

Morphology  and  Staining. — The  morphologic  characters  are 
practically  identical  with  those  of  B.  avisepticus,  as  are  also  the 
staining  characteristics. 

Isolation  and  Culture. — B.  suisepticus  may  be  isolated  in  pure 


Fig.  121. — Bacillus  suisepticus  (after 
deSchweinitz  and  McFarland). 


300  VETERINARY   BACTERIOLOGY 

culture  from  the  lungs,  from  the  blood,  and  from  various  internal 
organs  of  the  body,  without  difficulty.  The  cultural  characters 
do  not  differ  from  those  described  for  B.  avisepticus. 

Physiology. — The  physiologic  characters  do  not  differ  from  those 
of  the  group. 

Pathogenesis. — Experimental  Evidence. — Inoculation  of  the 
mouse,  rabbit,  and  fowl  lead  to  the  same  results  as  with  the  bacil- 
lus of  fowl  cholera.  Hogs  die  of  septicemia  after  subcutaneous 
injection.  It  has  not  proved  generally  possible  to  infect  the  hog 
by  feeding.  That  the  organism  is,  under  proper  conditions, 

pathogenic  for  the  hog  appears  to  be  well 
-  demonstrated,  but  that  it  can  produce 

' 

an  epizootic  naturally  among  animals  is 
kv  no  means  so  well  established. 
(J  Character    of     Disease    and     Lesions 

Produced. — The  characteristic  lesions  of 
Q)  this  disease  are  generally  to  be  found  in 

Fig.  l22.-Bacillus  sui-  the  lungs'  althouSh  the  intestines  may 

septicus   in    blood  (after  exhibit    some  changes  and   may  closely 

deSchweinitz,  Report  Bu-  simulate   the    conditions   found   in   hog- 

reau  of  Animal  Industry).  cholera      The  ^^  may>  fcherefor6j  be 

denominated  a  pneumoenteritis.     It  may 

also  appear  in  septicemia  form.  Punctiform  hemorrhages  are 
generally  to  be  observed,  particularly  in  the  kidneys. 

Immunity. — As  with  the  fowl-cholera  bacillus,  no  true  toxins 
have  been  demonstrated.  Both  active  and  passive  immunization 
against  the  B.  suisepticus  have  been  accomplished.  Active  im- 
munization has  been  attempted  in*  many  different  ways.  The  use 
of  killed  and  living  cultures  has  not,  in  general,  proved  satis- 
factory in  immunizing  the  hog,  although  they  have  been  success- 
fully used  in  the  preparation  of  antisera  from  the  horse  and  other 
animals.  Weil  has  elaborated  the  following  technic,  making  uso 
of  the  so-called  "  natural  aggressins  "  for  the  establishment  of 
immunity.  A  rabbit  is  injected  intraperitoneally  with  5  c.c.  of 
bouillon  containing  a  drop  of  twenty-four-hour  culture  of  a  highly 
virulent  strain  of  the  organism.  The  animal  should  die  within 
the  next  twenty-four  hours.  The  exudate,  varying  in  amount  from 
1  to  20  c.c.,  is  pipetted  off  and  sterilized  by  the  addition  of  0.5 


HEMORRHAGIC   SEPTICEMIA   GROUP  301 

per  cent,  of  phenol,  then  heated  to  44°  for  three  hours,  then  its 
sterility  determined  by  transfers  to  broth.  If  the  broth  shows  no 
growth,  the  material  is  sterile  and  is  ready  for  use.  This  may 
be  used  to  inject  laboratory  animals  and  thereby  establish  im- 
munity. The  animal  immediately  after  injection  becomes  more 
susceptible  to  the  disease,  presumably  due  to  the  presence  of  ag- 
gressin  in  the  blood,  but  later  a  relatively  permanent  active  im- 
munity is  produced.  In  practice  it  is  found  that  in  the  immuniza- 
tion of  hogs  it  is  necessary  that  the  exudate  containing  the  ag- 
gressin  be  obtained  from  other  hogs  rather  than  rabbits.  Wasser- 
mann  and  Citron  have  developed  a  somewhat  similar  method  of 
immunization  by  the  use  of  so-called  "  artificial  aggressins  "  or 
bacterial  extracts.  These  methods  of  immunization  are  of  much 
more  theoretic  than  practical  importance. 

Passive  immunization  by  means  of  antisera  has  been  studied 
by  several  investigators.  A  rabbit  may  be  actively  immunized 
by  one  of  the  preceding  methods,  and  its  serum  may  protect  a 
mouse  in  doses  of  less  than  0.1  c.c.  against  a  fatal  injection  of  a 
highly  virulent  organism.  Wassermann  and  Ostertag  and  their 
pupils  have  shown  that  an  antiserum  specific  for  one  strain  of 
B.  suisepticus  is  not  always  effective  for  others.  They,  therefore, 
prepare  serum  by  the  systematic  immunization  of  a  horse  against 
several  strains  of  the  organism  until  a  serum  of  high  potency 
is  produced.  Its  strength  is  determined  by  injections  into  mice. 
Experiments  upon  young  pigs  with  this  serum  are  claimed  to  have 
been  highly  successful,  but  the  method  has  not  come  into  general 
use.  Simultaneous  injections  of  immune  sera  and  of  B.  suisepti- 
cus have  also  been  advocated. 

In  summary  it  may  be  said  that  immunization  against  swine 
plague  is  still  in  the  experimental  stage,  and  that  no  completely 
satisfactory  method  has  been  evolved. 

Bacteriologic  Diagnosis. — The  identification  of  the  causal 
organism  by  actual  isolation  is  the  only  practicable  method  of 
bacteriologic  diagnosis. 

Transmission. — The  means  by  which  the  disease  spreads 
naturally  are  not  fully  understood.  It  is  possible  that  it  is  by 
ingestion,  prbbably  sometimes  by  inhalation. 


302  VETERINARY   BACTERIOLOGY 

Bacillus  bovisepticus 

Synonyms. — Bacterium  bovisepticum ;  Bacterium  bipolare  multi- 
cidum;  Bacillus  bovicida. 

Disease  Produced. — Hemorrhagic  septicemia  in  cattle,  buffalo, 
and  related  wild  animals;  Rinderseuche;  Wildseuche. 

The  early  descriptions  of  this  disease  refer  to  it  as  attacking 
cattle,  wild  animals,  and  swine.  Bellinger,  in  1878,  first  described 
it  as  Wild-  and  Rinderseuche,  attacking  wild  boars  and  deer. 
Kitt,  in  1885,  isolated  an  organism  belonging  to  the  hemorrhagic 
septicemia  group.  Since  that  time  numerous  investigators  have 
reported  epidemics  of  the  disease  in  many  countries.  Fernmore, 
in  1898,  first  noted  its  presence  in  the  United  States.  Wilson  and 
Bumhall,  in  1901,  and  Reynolds,  in  1903,  studied  several  epi- 
demics in  the  State  of  Minnesota. 

Morphology  and  Staining. — It  does  not  differ  from  the  B. 
avisepticus  and  B.  suisepticus. 

Isolation  and  Culture. — The  specific  organism  may  be  isolated 
from  the  blood  and  the  internal  organs  of  infected  animals.  The 
cultural  characters  are  practically  identical  with  the  preceding 
forms. 

Physiology. — Same  as  B.  avisepticus  and  B.  suisepticus. 

Pathogenesis. — Experimental  Evidence. — The  organism  is  path- 
ogenic for  the  mouse,  rabbit,  and  pigeon.  Inoculation  of  virulent 
cultures  into  cattle  have  been  successful  in  causing  the  disease. 

Character  of  Disease  and  Lesions  Produced. — The  presence 
of  small  hemorrhages  in  many  of  the  body  organs,  and  particularly 
upon  the  serous  surfaces,  is  characteristic.  Hemorrhages  are 
quite  uniformly  present  also  in  the  subcutaneous  tissues.  In 
some  cases  these  are  quite  extensive  and  involve  a  considerable 
portion  of  the  body  surface.  The  heart  is  usually  petechiated. 

Immunity. — It  is  entirely  prpbable  that  the  facts  relative  to 
immunity  given  under  the  discussion  of  fowl  cholera  and  swine 
plague  will  hold  good  in  bovine  hemorrhagic  septicemia.  How- 
ever, no  practical  method  of  immunization  is  known  and  little 
work  has  been  done  on  this  topic. 

Bacteriologic  Diagnosis.— Stained  mounts  from  the  blood  and 
the  internal  organs  will  show  the  gram-negativo,  polar-staining 
bacillus.  It  may  be  isolated  upon  culture-media.  Diagnosis 


HEMORRHAGIC   SEPTICEMIA   GROUP  303 

by  agglutination  may  be  practicable,  but  further  work  is  needed 
before  its  usefulness  can  be  ascertained. 

Transmission. — The  means  by  which  the  organism  spreads 
from  one  animal  to  another,  and  by  which  it  gains  entrance  to  the 
body,  is  not  well  understood. 

Other  Hemorrhagic  Septicemias  of  Animals 

Organisms  belonging  to  this  group  have  been  isolated  from  a 
considerable  number  of  other  animal  diseases  in  addition  to  the 
ones  which  have  already  been  described.  Rabbit  septicemia  or 
rabbit  plague  (Bacillus  cuniculicida) ,  pneumoenteritis,  or  hemor- 
rhagic  septicemia  of  sheep  and  of  the  horse,  infectious  pneumonia 
of  goats,  Biiffelseuche  or  pasteurellosis  of  the  buffalo,  dog  typhoid 
or  dog  pasteurellosis,  hemorrhagic  septicemia  of  elephants,  of 
geese,  wild  birds,  and  many  other  animals  have  been  ascribed  to 
Bacillus  septicemice  hcemorrhagicce.  As  has  been  before  stated,  the 
evidence  in  some  of  these  cases  seems  to  be  inconclusive. 

Bacillus  pestis 

Synonyms. — Bacterium  pestis;  B.  pestis  bubonicce. 

Disease  Produced. — Bubonic  plague  in  man  and  rodents. 

Yersin  and  Kitasato,  in  1894,  independently  described  the  or- 
ganism which  causes  bubonic  plague.  Since  that  time  it  has  been 
isolated  and  described  by  many  observers  in  numerous  outbreaks. 

Distribution. — The  disease  is  endemic  in  parts  of  China  and 
India.  At  various  times  it  has  spread  as  an  epidemic  over  the 
entire  civilized  world.  Cases  have  been  reported  within  recent 
years  in  most  of  the  civilized  countries,  but  it  has  not  gained  a 
foothold  and  spread  in  any  countries  except  those  of  southern 
Asia  and  China. 

Morphology  and  Staining. — The  Bacillus  pestis  morphologi- 
cally resembles  the  other  members  of  this  group.  It  is  small— 
usually  about  0.5  to  0.75  by  1.5  to  2  it.  It  sometimes  occurs  in 
short  chains,  but  is  usually  single.  Involution  forms  are  produced 
so  readily  upon  appropriate  culture-media,  such  as  partially 
desiccated  agar  and  salt  agar,  that  their  development  has  been 
regarded  as  diagnostic.  Capsules  may  sometimes  be  demonstrated 
on  culture-media,  but  not  in  tissues.  The  bipolar  staining  of 


304  VETERINARY   BACTERIOLOGY 

the  organism  is  particularly  evident  in  smears  from  tissues  or 
blood. 

Isolation  and  Culture. — The  organism  may  be  isolated  directly 
from  infected  lymph-glands  in  pure  culture.  It  has  been  ob- 
tained from  the  blood,  but  this  is  not  usual.  Growth  occurs 
readily  on  most  culture-media.  The  colonies  on  agar  are  delicate, 
drop-like,  with  center  somewhat  granular  and  thicker  than  the 
uneven,  slightly  granular  margin.  Growth  on  other  media  does 
not  differ  markedly  from  that  described  for  other  members  of  this 
group.  Growth  occurs  scantily  or  not  at  all  on  potato.  One 
character  that  has  been  described  and  has  been  found  useful  in 


Fig.  123.— Bacillus  peslis  (Wherry). 

diagnosis  is  the  "  stalactite  "  formation  in  broth  covered  with 
oil  and  allowed  to  remain  without  being  disturbed.  Under  these 
conditions  long  delicate  threads  are  produced  which  hang  from  the 
oil  and  resemble  the  stalactites  found  in  caves. 

Physiology. — The  organism  is  aerobic  and  facultative  anaerobic. 
The  optimum  growth  temperature  is  between  25°  and  30°,  but 
growth  occurs  both  above  and  below  these  temperatures.  It  is 
easily  destroyed  by  desiccation,  heat,  light,  and  disinfectants. 
Dextrose  broth  is  acidified,  but  no  gas  is  produced.  Indol  is  not 
formed. 


HEMORRHAGIC    SEPTICEMIA   GROUP  305 

Pathogenesis. — Experimental  Evidence. — The  organism  readily 
infects  mice,  rats,  guinea-pigs,  rabbits,  dogs,  cats,  and  monkeys, 
when  they  are  experimentally  inoculated.  The  symptoms  and 
lesions  produced  are  entirely  typical  of  bubonic  plague  in  man. 
Accidental  infection  of  man  resulting  in  four  cases  of  plague 
occurred  in  a  Vienna  laboratory  at  a  time  when  bubonic  plague  did 
not  exist  elsewhere  in  Europe.  The  causal  relationship  of  the 
organism  to  the  disease  may  be  held  to  be  fully  established. 

Character  of  Disease  and  Lesions  Produced. — The  disease  in 
experimental  animals  may  be  a  rapidly  fatal  septicemia,  or  in 
those  animals  which  are  somewhat  resistant,  as  the  rat,  typical 
buboes  (enlarged  and  suppurating  lymph-nodes)  or  abscesses 


**  •- 


Fig.  124. — Bacillus  pestis,  bacilli  from  a  bubo  (Giinther). 

in  the  spleen  or  liver  are  produced.  The  disease  in  man  may  be 
of  one  of  three  types — septicemic,  usually  rapidly  fatal  with  the 
organisms  generally  distributed  through  the  blood  and  various 
tissues  of  the  body;  the  pneumonic,  also  rapidly  fatal;  and  the 
bubonic,  the  most  common,  in  which  the  lymph-nodes  are  infected, 
become  enlarged,  and  ulcerate.  The  bubonic  type  is  less  fatal, 
recovery  taking  place  in  a  small  percentage  of  cases.  The  sep- 
ticemic type  of  the  disease  is  often  accompanied  by  extensive  sub- 
cutaneous hemorrhages,  which  gave  the  name  "  black  death  " 
to  the  epidemics  of  mediaeval  Europe. 

Immunity. — No  true  toxin  has  been  demonstrated  for  B.  pestis, 
although  endotoxins  have  been  shown  to  be  present.     Agglutinins 
20 


306  VETERINARY  BACTERIOLOGY 

for  this  organism  may  be  found  in  the  blood  of  advanced  cases 
and  of  convalescents,  but  they  appear  too  late  to  be  of  any  diag- 
nostic value.  The  reaction  is  rather  doubtfully  specific.  The 
reaction  occurs  only  in  low  dilutions — rarely  above  1 :  20.  Agglu- 
tination in  much  higher  dilutions  may  be  secured  with  immune 
serum — sometimes  as  high  as  1 :  1000  has  been  observed.  Pre- 
cipitins  have  also  been  noted  in  laboratory  experimentation. 
Opsonins  for  B.  pestis  have  been  demonstrated  in  normal  human 
serum  and  in  immune  serum.  Bactericidal  substances  are  pres- 
ent in  the  serum  of  artificially  immunized  animals. 

Active  immunization  of  man  against  bubonic  plague  has  been 
quite  extensively  practised.  The  procedure  consists  in  every 
case  of  injection  of  killed  or  attenuated  bacilli  or  their  products 
as  a  prophylactic  measure.  The  use  of  the  various  substances  has 
proved  quite  successful.  Haffkine's  vaccine  has  been  used  in 
India.  It  consists  of  a  killed  six-weeks  culture  of  plague  bacilli 
in  broth.  Modifications  of  this  method  have  been  utilized  by 
many  investigators.  The  immunity  established  is  probably  both 
opsonic  and  bactericidal. 

Passive  immunization  by  the  injection  of  the  serum  of  horses 
hyperimmunized  against  B.  pestis  has  been  highly  successful 
according  to  some,  and  of  no  material  advantage  according  to 
others.  Several  procedures  have  been  advocated.  The  following 
is  that  of  Kolle  and  Kumbein:  A  culture  of  B.  pestis  is  passed 
through  rats  to  exalt  its  virulence.  This  is  planted  upon  agar  and 
incubated  forty-eight  hours  at  30°,  the  growth  washed  off  and  sus- 
pended in  physiological  salt  solution.  The  suspension  is  killed  by 
heating  to  70°  for  an  hour  and  its  sterility  determined.  The 
horse  is  injected  at  intervals  of  a  few  days  with  gradually  increasing 
doses  of  the  dead  bacteria,  until  after  seven  or  eight  injections  the 
bacteria  from  six  or  more  culture-tubes  are  injected  at  one  time. 
Injections  of  minute  quantities  of  the  living  organism  are  then 
begun  and  finally  after  repeated  injections  the  living  organisms 
from  16  cultures  are  used.  The  interval  between  injections  is 
governed  by  the  reaction  of  the  animal.  Usually  it  is  from  five 
to  eight  days.  The  animal  is  then  bled,  and  the  serum  preserved 
by  the  addition  of  0.5  per  cent,  phenol. 

Bacteriologic   Diagnosis. — The  organism  may  be    recognized 


HEMORRHAGIC    SEPTICEMIA   GROUP  307 

in  stained  mounts  from  the  pus  from  a  bubo  as  a  small  gram- 
negative  bacillus,  with  characteristic  bipolar  staining.  It  may 
also  be  isolated  upon  culture-media  and  identified  by  its  growth 
characteristics. 

Transmission. — The  pneumonic  form  of  the  disease  may  be 
transmitted  by  the  inhalation  of  infectious  droplets.  Plague  is 
not  known  to  occur  in  the  human  following  ingestion  of  the  or- 
ganism. The  bubonic  or  most  common  type  is  probably  trans- 
mitted to  man  by  the  bite  of  fleas  (or  from  their  excretions  scratched 
into  the  skin),  which  have  left  rats  dead  of  the  disease.  An  epi- 
demic of  plague  in  the  human  is  commonly  preceded  by  an  epizo- 
otic among  the  rats  of  the  community.  It  has  been  shown  ex- 
perimentally that  a  flea  may  transmit  the  disease  from  an  infected 
to  a  non-infected  individual.  It  is  also  known  that  the  cannibal- 
istic tendency  of  the  rats  to  eat  their  dead  is  in  part  responsible 
for  the  spread  of  the  disease  among  vermin.  The  annihilation 
of  the  rat  is  the  best  prophylaxis  known.  The  disease  has  in 
certain  places,  as  about  San  Francisco,  been  found  to  spread  to 
such  rodents  as  the  ground-squirrels  and  wood-rats.  When  a 
region  once  becomes  thoroughly  infected,  it  is,  therefore,  difficult 
to  stamp  out  the  disease. 


CHAPTER  XXX 

ACID-FAST  GROUP 

THE  Bacillus  tuberculosis,  B.  kprce,  the  bacillus  of  Johnes' 
disease,  and  certain  common  non-pathogenic  bacteria  isolated 
from  hay,  dung,  milk,  butter,  and  other  sources,  have  common 
morphological  and  staining  characters  which  associate  them  as  the 
acid-fast  group.  The  crucial  test  for  these  forms  is  their  ability 
to  retain  stains  when  treated  with  strong  acids.  They  do  not 
stain  readily  with  the  common  anilin  dyes,  but  when  once  stained 
they  are  "  acid-fast."  Hot  carbol-fuchsin  is  commonly  used  in 
determining  this  character. 

The  members  of  the  acid-fast  group  of  bacteria  are  all  slender, 
non-motile  rods,  without  capsules  or  spores,  gram-positive,  and 
acid-fast.  Slight  variations  in  morphology  and  cultural  characters 
and  considerable  differences  in  pathogenesis  serve  to  differentiate 
the  various  species.  All  the  species  may  occasionally  produce 
branched  cells  and  threads,  and  it  has  been  concluded  by  some  in- 
vestigators that  they  belong  rather  with  the  fungi,  or  at  least  with 
the  higher  bacteria  than  with  the  lower  bacteria. 

Care  should  be  used  not  to  confuse  the  term  pseudotuberculosis, 
and  particularly  the  bacteria  associated  with  that  disease,  with  the 
true  tubercle  bacilli  and  related  forms.  As  has  before  been  stated, 
the  term  pseudotuberculosis  is  purely  pathological,  and  refers  to 
the  type  of  lesions  produced  in  the  body,  and  not  to  any  resemblance 
of  the  organisms  causing  the  disease  to  those  of  tuberculosis. 
The  pseudotubercle  bacilli  are  more  closely  related  to  the  diph- 
theria group,  and  have  already  been  discussed. 

Bacillus  tuberculosis 

Synonyms. — Bacterium  tuberculosis;  Mycobacterium  tubercu- 
losis. 

Disease  Produced. — Tuberculosis  in  mammals  and  birds. 
806 


ACID-FAST   GROUP  309 

The  disease  in  its  various  forms  in  man  and  animals  has  been 
known  since  ancient  times.  Villemin,  in  1865,  showed  that  the 
disease  could  be  transferred  from  one  animal  to  another  by  injec- 
tion of  the  crushed  nodules.  He  did  not  succeed  in  discovering  the 
specific  organism,  however.  In  1884  Dr.  Robert  Koch  presented 
the  results  of  his  studies  of  the  disease  and  described  the  organism 
which  he  had  proved  to  be  its  etiological  factor.  This  work  will 
always  stand  as  a  model  of  scientific  research  in  bacteriology 
carried  out  under  the  most  difficult  circumstances.  Staining 
methods  and  culture-media  were  both  devised  before  the  organism 
could  be  seen  or  grown.  Koch  succeeded  in  demonstrating  its 
presence  in  the  characteristic  lesions,  in  growing  it  upon  artificial 
media,  and  in  reproducing  the  disease  in  animals  by  the  inocula- 
tion of  pure  cultures.  It  was  assumed  in  the  beginning  that  tuber- 
culosis in  all  animals  was  caused  by  the  same  organism,  but  in  1896 
Theobald  Smith  called  attention  to  the  fact  that  certain  differ- 
ences in  cultural,  morphological,  and  pathogenic  characters  could  be 
distinguished  in  the  organisms  isolated  from  human  and  bovine 
tuberculosis.  Koch,  in  1901,  declared  the  two  organisms  to  be 
distinct,  and  that  the  probability  of  human  infection  with  bovine 
tuberculosis  was  remote  indeed.  Since  that  time  the  subject  has 
been  studied  with  varied  results  by  many  investigators.  Prob- 
ably more  has  been  written  on  this  one  disease  than  any  ten  or 
more  other  diseases.  Journals  devoted  exclusively  to  tuberculosis 
are  issued.  Many  points  even  yet  are  not  fully  understood,  but 
the  subject  is  upon  a  moderately  sound  basis  at  present.  It  seems 
best  to  differentiate  three  varieties  of  the  tubercle  bacillus — the 
human,  the  bovine,  and  the  avian.  These  possess  many  points 
in  common,  and  it  is  by  no  means  certain  that  they  cannot  be 
transformed  the  one  into  the  other,  but  they  may,  in  general,  be 
readily  differentiated  by  specific  morphological  and  physiological 
characters.  They  will  be  discussed  together. 

Distribution. — Tuberculosis  in  man  is  known  throughout  the 
civilized  world.  In  the  United  States  over  110,000  deaths  occur 
annually  from  this  disease.  It  usually  leads  all  other  diseases  in 
total  number  of  deaths  produced.  The  disease  in  cattle  is  nearly 
as  widely  distributed.  It  is  found  throughout  Europe  and  America. 
Isolated  localities  free  from  the  disease,  however,  are  known. 


310 


VETERINARY   BACTERIOLOGY 


In  the  United  States  it  is  rarely  encountered  on  the  western  ranges, 
but  is  relatively  common  in  dairy  and  beef  herds  in  other  parts 
of  the  country.  Whenever  swine  are  allowed  to  follow  tuberculous 
cattle,  they  commonly  become  infected.  The  same  is  also  true 
when  they  are  fed  upon  unpasteurized  milk  from  tuberculous 
animals.  Tuberculosis  occurs  rarely  in  the  horse.  Sheep  have 
been  reported  as  tuberculous,  but  the  disease  is  certainly  rare;  it 
is  probably  frequently  confused  with  pseudotuberculosis  or  caseous 
lymphadenitis  in  this  animal.  Tuberculosis  in  domestic  fowls  is 
known  to  occur  in  many  European  localities,  and  in  the  United 
States  has  been  reported  from  Oregon,  California,  and  New  York. 


. 

• 


I 


Fig.  125.  —  Bacillus  tuberculosis  in 
human  sputum.  Note  the  slender 
beaded  character  of  the  rods  (X 
1000)  (Giintherj. 


Fig.  126.  —  Bacillus  tuberculosis, 
human,  mount  from  glycerin  agar 
(Frankel  and  Pfeiffer). 


Morphology  and  Staining.  —  B.  tuberculosis  is  a  slender  rod, 
frequently  somewhat  bent,  with  rounded  ends.  It  varies  from 
0.2  to  0.5  by  1.5  to  3.5  ^,  and  sometimes  longer.  Frequently  the 
protoplasm  takes  the  stain  irregularly  and  gives  a  beaded  appear- 
ance to  the  cell.  No  spores  or  capsules  are  produced.  The  organ- 
ism is  non-motile.  Branched  and  elongated  forms  resembling 
somewhat  the  actinomyces  are  sometimes  observed.  It  is  probable 
that  these  are  involution  forms,  although  some  authors  chiim  I  linn 
to  be  developmental  forms  instead.  The  organism  stains  with 
difficulty,  but  when  once  stained,  is  acid-fast.  It  is  possible  that 


ACID-FAST   GROUP 


311 


under  certain  conditions,  in  the  animal  tissues  in  particular,  this 
acid-fast  property  may  be  temporarily  lost.  The  acid-fast  char- 
acter is  apparently  due  to  the  presence  of  a  wax-like  substance  in 
the  bacterial  cell.  The  cells  from  which  this  has  been  removed  by 
ether  and  benzol  are  no  longer  acid-fast. 

There  are  certain  morphological  differences  commonly  to  be 
observed  between  bovine  and  human  tubercle  bacilli.  The 
former  are  shorter,  straighter,  and  thicker  than  the  latter.  They 
are  also  less  apt  to  show  the  irregular  or  granular  staining  noted 
above.  These  characters  are,  of  course,  not  sufficient  to  differen- 
tiate isolated  bacteria  of  the  two  types,  but  cultures  can  usually 
be  identified  readily  by  an  ex- 
perienced observer.  Whether 
or  not  one  type  may  be  trans- 
formed into  the  other  type 
by  animal  inoculation  or  by 
cultural  methods  is  a  moot 
question.  Some  investigators 
claim  to  have  isolated  typical 
human  bacilli  from  animals 
that  have  been  inoculated 
with  bovine  bacilli,  others 
hold  that  there  is  no  evidence 
of  the  transformation  of  the 
one  type  to  the  other.  How- 
ever this  may  ultimately  be 
decided,  it  is  certain  that  each 

type  retains  its  characters  with  a  considerable  degree  of  con- 
stancy. The  existence  of  two  well-marked  varieties  is  unques- 
tioned. The  bacillus  of  avian  tuberculosis  resembles  the  bovine 
type  closely  in  its  morphology  and  staining  reactions. 

Isolation. — The  isolation  of  B.  tuberculosis  from  lesions  is 
attended  with  considerable  difficulty.  This  is  even  more  pro- 
nounced when  an  attempt  is  made  to  secure  the  organism  from  the 
sputum  or  the  feces  where  it  exists  in  mixed  culture. 

It  may  be  isolated  from  infected  organs  by  securing  bits  of  the 
tissue  and  rubbing  over  the  surface  of  inspissated  blood-serum  or 
other  suitable  medium.  The  method  worked  out  by  Theobald 


Fig.  127. — Bacillus  tuberculosis,  bo- 
vine, in  a  section  of  the  peritoneum 
(Frankel  and  Pfeiffer). 


312  VETERINARY  BACTERIOLOGY 

Smith  has  given  excellent  results  in  the  hands  of  numerous  in- 
vestigators. A  dog  is  bled,  using  all  aseptic  precautions,  from  the 
femoral  artery  into  a  sterile  vessel  and  the  blood  allowed  to  clot. 
The  serum  is  removed  by  sterile  pipettes  to  sterile  test-tubes. 
These  are  slanted  and  heated  to  a  temperature  of  75°  to  76°  for 
about  three  hours,  or  until  the  serum  is  coagulated.  The  heating 
must  be  done  in  a  saturated  atmosphere  and  the  medium  stored  so 
that  there  is  no  loss  by  evaporation.  Bits  of  infected  tissue  are 
placed  upon  the  surface  and  kept  in  a  thermostat  for  several  weeks. 
If  no  growth  appears,  the  tissue  is  moved  about  and  incubated 
again.  A  constant  temperature  of  37  °  and  a  saturated  atmosphere 
must  be  maintained. 

A  procedure  somewhat  simpler  than  the  preceding  has  been 
described  by  Dorset  and  is  found  to  give  good"  results.  The  shell 
of  fresh  eggs  is  carefully  broken,  and  the  white  and  yolk  dropped 
into  a  sterile  flask,  the  yolk  broken  with  a  sterile  rod  or  wire,  and 
the  contents  of  the  flask  shaken  until  the  two  are  thoroughly  mixed. 
Foaming  is  to  be  avoided.  The  mixture  is  placed  in  tubes,  slanted, 
and  heated  at  a  temperature  of  about  70°  for  from  four  to  five  hours 
on  two  days.  This  coagulates  and  sterilizes  the  medium.  The 
tubes  should  be  stored  where  they  will  not  lose  water  by  evapora- 
tion. Several  drops  of  distilled  sterile  water  should  be  added  to  a 
tube  just  before  incubation.  The  isolation  upon  this  medium  is 
carried  out  as  outlined  above.  A  growth  may  generally  be  ob- 
served within  ten  days  after  inoculation  with  fresh  tissue. 

Isolations  from  sputum  or  feces,  milk,  or  other  substances  in 
which  the  organisms  occur  mixed  with  other  forms,  is  attended  with 
some  difficulty.  It  is  usually  accomplished  by  injecting  the  mate- 
rial or  the  sediment  yielded  by  centrifugation  directly  into  a  guinea- 
pig.  The  bacilli  may  later  be  isolated  in  pure  culture  from  the 
nodules  produced.  Within  recent  years  the  use  of  "  antiformin  " 
and  similar  substances  has  considerably  simplified  this  procedure. 
Antiformin  is  the  trade-name  given  a  disinfectant  mixture  having 
the  following  composition: 

Solution  I.     Sodium  carbonate 12  gm. 

Chlorinated  limo 8  RMI. 

Distilled  water SO  gin. 

Solution  II.  Sodium  hydroxid 15  gm. 

Distilled  water 85  gm. 


ACID-FAST   GROUP  313 

Equal  quantities  of  the  two  solutions  are  mixed  for  use.  The 
sputum  or  other  material  containing  the  tubercle  bacilli  is  placed 
in  a  centrifuge  tube,  and  antiformin  to  about  20  per  cent,  of  its 
bulk,  added.  The  tube  is  then  corked,  thoroughly  shaken,  and  al- 
lowed to  remain  in  a  dark  place  for  twenty-four  hours.  It  is  then 
centrifuged,  the  clear,  supernatant  liquid  pipetted  off,  the  tube 
filled  with  sterile  physiological  salt  solution,  centrifuged,  washed 
a  second  time,  and  the  sediment  smeared  over  the  surface  of  serum 
slants.  The  antiformin  destroys  all  other  non-acid-fast  bacteria 
present  and  dissolves  the  mucus  and  most  of  the  cell  elements, 
but  when  properly  used,  seems  to  have  little  effect  upon  the  tuber- 
cle bacilli,  as  they  retain  their  vitality  unimpaired.  This  is  prob- 
ably because  of  their  chemical  composition  and  waxy  covering. 

Cultural  Characters. — B.  tuberculosis  does  not  grow  readily 
when  first  isolated  upon  culture-media,  and  will  grow  only  upon 
certain  substances.  After  a  few  transfers  it  seems  to  become  ha- 
bituated to  growth  under  these  conditions  and  will  develop  through 
a  much  greater  range  of  temperature  and  on  other  media.  Devel- 
opment occurs  best  on  media  containing  blood-serum,  egg,  or 
similar  proteins,  or  to  which  glycerin  has  been  added. 

The  colonies  upon  blood-serum  or  glycerin  agar  appear  in  the 
course  of  ten  days  or  two  weeks  as  tiny  grains  barely  visible  to  the 
naked  eye.  They  gradually  enlarge,  and  in  subcultures  become 
confluent  and  cover  the  surface  of  the  medium  with  a  dry,  rather 
mealy,  wrinkled  growth;  the  colonies  direct  from  lesions  do  not 
coalesce  usually.  The  growth  is  white  and  lusterless,  or  rarely  in 
old  cultures  cream  or  brown.  In  glycerin  bouillon  the  growth 
generally  occurs  as  a  more  or  less  continuous,  heavy,  wrinkled, 
white  pellicle  that  breaks  into  pieces  and  sinks  to  the  bottom  when 
the  medium  is  shaken.  Similar  growths  occur  upon  other  media 
which  contain  glycerin.  In  no  other  case  is  the  growth  so  rapid. 

Cultural  characters  which  may  be  used  in  the  certain  differen- 
tiation of  the  human,  bovine,  and  avian  tubercle  bacilli  are  not 
readily  found.  The  organisms  isolated  from  the  human  and  from 
the  bird  adapt  themselves  much  more  readily  to  artificial  media 
and  grow  more  luxuriantly  than  does  the  bovine  type. 

Physiology. — The  B.  tuberculosis  is  aerobic.  Its  optimum 
growth  temperature  is  about  37.5°  for  the  human  and  the  bovine 


314 


VETERINARY    BACTERIOLOGY 


types  and  somewhat  higher  for  the  avian.  The  thermal  death- 
point  is  60°  for  twenty  minutes.  This  is,  therefore,  the  minimum 
time  and  temperature  for  the  efficient 
pasteurization  of  milk.  Sunlight  de- 
stroys the  organism  quickly,  but  it  is 
moderately  resistant  to  desiccation. 
Most  of  the  physiological  characters, 
such  as  acid,  gas,  pigment, '  and  indol 
production,  are  negative. 

Theobald  Smith  has  called  attention 
to  what  appears  to  be  a  very  constant 
differential  character  between  human  and 
bovine  tubercle  bacilli.  He  found  that 
in  glycerin  bouillon  (2  per  cent.)  acid  to 
phenolphthalein  the  human  bacillus 
causes  a  permanent  acid  reaction,  while 
with  the  bacillus  of  bovine  origin  the 
acidity  diminishes  and  the  reaction  be- 
comes alkaline  if  the  growth  environ- 
ment of  the  culture  is  suitable.  The 
tuberculin  prepared  from  the  human 
bacillus  is  acid,  and  from  the  bovine 
bacillus  alkaline.  This  difference  has 
been  noted  by  other  investigators  since 
its  first  description,  and  seems  to  be  one 
of  the  best  methods  of  differential  diag- 
nosis. 

Pathogenesis. — Tuberculosis  is  char- 
acteristically a  chronic  disease.  Even 
in  experimental  animals  months  are  often 
required  for  it  to  run  its  course.  As 
stated  by  Moore,  "It  does  not  destroy 
life  by  acute  toxemia,  but  by  a  chronic 
and  long-continued  systemic  poisoning 
and  by  the  morbid  changes  brought  about 
through  the  localization  of  these  lesions 
in  the  organs  necessary  to  life." 
Experimental  Evidence  of  Pathogenesis. — The  laboratory  animals 


Fig.  128.  —  Bacillus 
tuberculosis,  glycerin  agar 
slant  (Curtis). 


ACID-FAST   GROUP 


315 


are  generally  susceptible  to  infection  with  B.  tuberculosis.  The 
constant  presence  of  the  organism  in  the  lesions  of  the  disease 
and  its  ability  to  reproduce  the  disease  are  sufficient  evidence 
that  it  is  the  true  etiologic  factor. 

Important  differences  in  pathogenesis  are  to  be  noted  among 
the  three  varieties.  The  bovine  bacillus  is  most  pathogenic  for 
laboratory  animals,  the  human  next,  and  the  avian  least  (except 


Fig.  129. — Tubercular  hypertrophy  of  the  intestinal   wall   in   the   bovine 

(Chausse). 

for  birds).  Guinea-pigs  inoculated  subcutaneously  with  bovine 
bacilli  generally  succumb  in  less  than  fifty  days;  those  inoculated 
with  human  bacilli  generally  live  more  than  fifty  days.  Intra- 
peritoneal  injection  of  the  bovine  type  is  fatal  in  seven  to  eighteen 
days,  of  the  human  type  in  from  ten  to  thirty-eight  days.  The 
difference  upon  intravenous  injection  of  the  rabbit  is  even  more 
marked — with  bovine  bacilli  death  occurs  within  three  weeks, 
with  human  bacilli  the  animals  usually  live  for  several  months 


316  VETERINARY   BACTERIOLOGY 

and  may  even  recover.  The  avian  type  ordinarily  does  not  pro- 
duce fatal  infection  in  guinea-pigs,  although  rabbits  succumb  and 
fowls  and  pigeons  contract  the  disease  readily. 

Character  of  Disease  and  Lesions  Produced. — Almost  any  part 
of  the  body  may  be  affected  with  tuberculosis.  The  disease, 
wherever  found,  generally  involves  the  lymphatics.  It  is  charac- 
terized by  the  development  of  nodules  having  an  essentially  similar 
structure  in  all  tissues.  The  presence  of  the  tubercle  bacilli  in  a 
tissue  causes  a  proliferation  of  the  fixed  connective-tissue  cells  to 
form  the  beginning  of  a  miliary  tubercle.  Lymphocytes  are  gener- 
ally attracted  and  are  present  in  the  surrounding  tissues  in  con- 
siderable numbers.  A  more  or  less  definite  layer  of  "  epithelioid  " 
cells  forms  the  boundary  of  the  tubercle.  Typical  giant-cells 
with  peripheral  nuclei  are  found  near  the  center.  Coagulation 
necrosis  proceeds  and  the  interior  caseates.  Encapsulation  with 
fibrous  tissue  may  occur,  and  the  whole  may  eventually  become 
calcified.  These  calcareous  grains  persist  in  healed  tuberculous 
areas.  Tubercles  frequently  are  formed  in  masses.  In  the  cow 
tubercular  lymph-glands  sometimes  become  as  large  or  larger  than 
an  orange. 

The  arrangement  of  the  nodules  frequently  shows  clearly  the 
path  of  the  spread  of  the  bacilli  through  lymphatic  metastases. 
Direct  growth  through  tissues  with  invasion  of  new  areas  probably 
rarely  occurs.  The  organisms  are  not  commonly  found  in  the 
blood-stream,  although  they  may  sometimes  be  carried  to  other 
parts  of  the  body  by  this  means.  Infection  of  the  bones,  joints, 
and  meninges  probably  occurs  in  this  manner. 

The  organs  most  commonly  the  seat  of  lesions  vary  with  the 
species  of  animal  and  the  mode  of  infection.  In  the  human,  pul- 
monary infection  (consumption)  is  most  common,  although  in- 
testinal tuberculosis,  infection  of  the  lymphatics  of  the  neck 
(scrofula) ,  of  the  bones  and  joints  (tubercular  osteitis  and  arthritis), 
of  the  meninges,  and  of  the  liver,  spleen,  kidneys,  and  other  organs 
of  the  body  and  the  serous  membranes  lining  the  cavities,  are  not 
uncommon.  Lupus  or  tuberculosis  of  the  skin  is  of  frequent 
occurrence  in  certain  European  countries.  Cattle  generally  show 
nodules  in  the  mesentery  and  in  the  peritoneum  (Perlsucht  or 
pearl  disease).  The  lungs  and  the  accompanying  lymph-glands 


ACID-FAST   GROUP 


317 


and  the  intestines  commonly  show  lesions.  In  a  certain  small 
percentage  of  tuberculous  cows,  variously  estimated  from  a  frac- 
tion of  1  to  5  per  cent.,  tuberculous  lesions  may  be  found  in  the 
udder.  Any  and  all  of  the  organs  of  the  body  may  be  infected. 
Swine  are  most  commonly  infected  in  the  lymph-glands  of  the 
neck  (swine  scrofula) ,  and  in  the  abdominal  organs  and  the  lungs. 
Avian  tuberculosis  most  frequently  attacks  the  abdominal  organs, 
particularly  the  liver  and  spleen,  more  rarely  the  lungs. 

Immunity. — No  true  toxin   has  been   demonstrated   for  the 
tubercle  bacillus.     Endotoxins  are  produced.     These  are  liberated 


i 


Fig.  130. — Section  of  a  tubercular  intestinal  wall  showing  the  bacilli  and  giant- 
cells  (Chausse). 

from  the  cell  with  difficulty  because  of  its  composition  and  slow 
dissolution.  Specific  agglutinins  and  precipitins  have  been  de- 
monstrated in  the  blood  of  infected  individuals  and  in  immune 
serum.  Opsonins,  both  normal  and  immune,  have  been  shown  to 
occur.  The  development  of  bacteriolysins  has  not  been  satis- 
factorily demonstrated. 

Methods  of  active  immunization  are  all  dependent  upon  the  use 
of  killed  or  attenuated  bacteria  or  their  products.  The  name  tuber- 
culin is  given  to  any  suspension  of  dead  tubercle  bacilli  or  a  solu- 


318 


VETERINARY   BACTERIOLOGY 


tion  of  their  products.  As  will  be  noted  later,  tuberculin  is  not 
only  of  therapeutic  significance,  but  of  great  diagnostic  value. 
Many  types  have  been  prepared  by  various  workers.  Some  of 
the  more  important  will  be  described  before  a  discussion  of  their 
use  in  immunization  and  in  diagnosis  is  undertaken. 


Fig.    131. — Tuberculin   flask,   showing  the  growth   of   Bacillus   tuberculosis 

(McFarland). 

Koch's  Old  Tuberculin  (Alt  Tuberculin)  .—Flat-bottomed 
flasks  containing  4  per  cent,  glycerin  broth  to  a  depth  of  2  to  3  cm. 
are  inoculated  with  B.  tuberculosis.  The  inoculated  material  is 
carefully  placed  on  the  glass  at  the  surface  of  the  liquid  in  order 
that  it  may  spread  out  at  once  as  a  film  over  the  surface.  Unless 
the  organism  is  at  the  surface,  little  or  no  growth  will  occur.  In 


ACID-FAST   GROUP  319 

the  course  of  four  weeks  at  blood-heat  the  surface  of  the  broth  is 
covered  with  a  heavy,  wrinkled  pellicle.  By  the  end  of  eight  weeks 
it  is  ready  for  the  preparation  of  the  tuberculin.  The  contents  of 
several  flasks  are  then  united  and  placed  in  a  porcelain  evaporating 
dish  on  a  water-bath.  The  material  is  concentrated  to  about 
one-tenth  of  its  original  bulk,  when  the  glycerin  content  becomes 
about  40  per  cent.  This  constitutes  the  tuberculin  commonlj'  used. 

Tuberculin  of  Denys. — This  investigator  believed  that  the 
efficiency  of  the  tuberculin  was  impaired  by  the  heat  used  in  con- 
centration. He  filtered  unheated  broth  cultures  through  porcelain 
and  utilized  the  filtrate. 

The  tuberculol  of  Landmann,  the  tuberculocidin  or  antiphthisin 
(A.  P.)  of  Klebs,  the  oxytuberculin  of  Hirschfelder  are  all  tuber- 
culins prepared  by  various  modifications  of  the  original  Koch 
method,  such  as  repeated  extraction  of  bacilli  at  different  tempera- 
tures, treatment  with  H2O2,  etc. 

Koch's  purified  tuberculin  is  prepared  by  adding  lj  volumes  of 
absolute  alcohol  to  the  crude  tuberculin  and  allowing  the  mixture 
to  stand  twenty-four  hours,  collecting  the  precipitate  on  a  filter 
and  washing  in  60  per  cent,  alcohol,  and  finally  drying  in  a  desic- 
cator at  100°.  This  material  is  diluted  in  water  or  glycerin  before 
use,  being  soluble  in  these. 

Tuberculin  R.  or  T.  R.  of  Koch. — Young  virulent  cultures  of 
tubercle  bacilli  are  dried  in  a  vacuum,  then  ground  in  an  agate 
mortar  or  ball  mill  for  a  considerable  period.  The  resultant  pow- 
der is  next  suspended  in  water,  shaken,  and  centrifuged  for  half 
to  three-quarters  of  an  hour  at  a  speed  of  4000  revolutions  per 
minute.  The  bacterial  fragments  are  thrown  to  the  bottom.  The 
portion  of  the  tubercle  bacilli  that  remain  in  solution  Koch  termed 
Tuberculinum  O.  (T.  O.);  the  portion  which  is  precipitated  was 
called  Tuberculinum  R.  (T.  R.).  Koch  dried  this  T.  R.,  reground 
it,  suspended  and  centrifuged  to  get  rid  of  all  the  T.  O.  The  T. 
R.  was  originally  planned  for  use  in  immunization.  This  residual 
material  was  found  to  be  largely  free  from  the  toxic  action  of  other 
tuberculin  and  of  the  T.  O.  This  preparation  has  not  come  into 
use,  inasmuch  as  there  is  always  a  possibility  that  living  virulent 
organisms  may  survive  the  treatment  and  produce  disease  when 
injected. 


320  VETERINARY   BACTERIOLOGY 

New  Tuberculin  or  Bacillus  Emulsion  of  Koch. — The  cultures 
are  prepared,  dried,  and  ground  as  for  the  manufacture  of  T.  R. 
They  are  then  suspended  in  glycerinated  physiologic  salt  solution. 
Some  preservative,  as  phenol,  is  added. 

This  by  no  means  completes  the  list  of  preparations  of  the 
tubercle  bacillus.  Most  of  them  are  of  laboratory  significance 
only,  the  ones  of  any  considerable  practical  importance  being  the 
old  tuberculin  and  the  purified  product. 

The  use  of  tuberculin  as  a  prophylactic  or  cure  has  not  proved 
successful  with  the  lower  animals,  nor  has  it  in  man  when  used 
in  large  doses.  Within  recent  years  it  has  come  into  common  use 
in  the  treatment  of  human  tuberculosis,  minute  injections  being 
given  and  care  taken  that  no  febrile  reaction  shall  follow.  Deter- 
minations of  the  opsonic  index  from  time  to  time  have  been  used 
with  success  in  the  determination  of  the  proper  spacing  of  the  in- 
jections. This  method  is  intended  to  stimulate  opsonin  produc- 
tion and  thus  aid  the  body  in  ridding  itself  of  the  bacilli. 

The  use  of  attenuated  cultures,  and  particularly  of  cultures  of 
the  B.  tuberculosis  from  the  human,  has  been  advocated  by  von 
Behring  and  others  as  a  practicable  vaccination  method  against 
tuberculosis  in  cattle.  This  method  seemed  to  promise  good 
results  at  first,  but  has  failed  when  tried  on  an  extensive  scale. 

Antisera  have  been  prepared  by  repeated  injections  of  various 
animals  with  tuberculin  T.  R.  and  other  products  of  the  tubercle 
bacillus.  None  of  them  has  proved  successful  in  conferring  passive 
immunity  on  other  individuals. 

In  summary  it  may  be  stated  that  up  to  the  present  time  no 
practicable  method  of  immunization  against  tuberculosis  in  cattle 
has  been  developed,  although  in  man  the  use  of  carefully  regulated 
injections  of  tuberculin  is  promising. 

Bacteriologic  Diagnosis. — Tuberculosis  may  be  diagnosed  bac- 
teriologically  by  staining  methods,  animal  inoculation,  agglutin- 
ation, and  the  tuberculin  reaction. 

Diagnosis  by  Staining  Methods. — Tubercle  bacilli  may  be 
readily  recognized  by  their  acid-fast  character  when  examined 
microscopically.  The  presence  of  the  characteristic  acid-fast 
bacteria  in  the  tissues  or  sputum  is  generally  sufficient  to  establish 
diagnosis.  This  is  not  the  case,  however,  with  feces,  milk,  or 


ACID-FAST   GROUP  321 

other  substances  which  may  become  contaminated  with  non- 
pathogenic  acid-fast  forms  common  in  dust,  in  soil,  etc.  Resort 
must  then  be  had  to  animal  inoculation  followed  by  isolation  of 
the  characteristic  bacillus,  or  to  isolation  by  the  use  of  antiformin. 
The  discovery  of  the  bacilli  in  milk,  sputum,  and  other  body 
secretions  may  frequently  be  greatly  facilitated  by  centrifugation 
and  by  the  preparation  of  mounts  from  the  sediment.  Anti- 
formin mixed  with  the  material  to  be  examined  greatly  aids  in  its 
sedimentation  without  interfering  in  any  way  with  its  staining 
properties,  providing  care  is  used  in  the  washing  as  described  under 
"  isolation." 

Diagnosis  by  Animal  Inoculation. — This  is  the  most  delicate 
method  of  determining  the  presence  of  tubercle  bacilli.  Intra- 
peritoneal  injections  of  the  suspected  material  into  a  guinea-pig 
will  result  in  the  development  of  the  disease  within  a  few  weeks. 
Non-pathogenic  acid-fast  bacteria  may  give  some  of  the  patho- 
logical appearances  of  true  tuberculosis,  so  that  it  is  well  to  make 
certain  of  the  diagnosis  by  isolation  and  cultivation  of  the  or- 
ganism from  the  inoculated  animal.  An  injection  of  tuberculin 
may  be  used  to  shorten  the  period  of  time  necessary  to  diagnosis 
in  the  inoculated  guinea-pig. 

Diagnosis  by  the  Agglutination  Test. — Although  specific  agglut- 
inins  for  the  tubercle  bacillus  may  be  demonstrated  in  the  blood 
of  those  having  the  disease,  the  diagnosis  by  this  method  has 
not  proved  practicable.  Great  care  is  necessary  to  secure  a  homo- 
geneous suspension  of  the  bacteria,  and  the  agglutinins  are  rarely 
present  in  quantity. 

Diagnosis  by  the  Tuberculin  Tests. — Subcutaneous  injection  of 
tuberculin  into  an  infected  animal  causes  a  characteristic  reaction. 
The  nature  of  this  reaction  varies  to  a  considerable  extent  with  the 
manner  and  site  of  the  injection  or  application.  Subcutaneous 
injections  are  usually  used  in  cattle;  in  man  the  dermo-,  cuti-,  or 
ophthalmo-reactions  are  commonly  employed. 

The  test  of  cattle  is  made  by  injecting  a  standard  dose  of  tuber- 
culin. This  is  about  the  equivalent  of  0.25  c.c.  of  Koch's  Old 
Tuberculin.  The  normal  temperature  of  the  animal  should  be 
ascertained  before  injection.  This  is  most  accurately  determined 
by  taking  the  temperature  every  two  hours  on  the  day  preceding 
21 


322  VETERINARY    BACTERIOLOGY 

the  test,  but  in  practice  frequently  but  one  or  two  preliminary 
determinations  are  made.  After  six  or  eight  hours  the  tempera- 
ture is  again  to  be  taken  every  two  hours  for  the  remainder  of  the 
twenty-four  hours  after  injection.  Care  must  be  exercised  that 
the  animals  are  kept  under  normal  conditions  during  the  test,  and 
that  they  remain  quiet.  Animals  in  heat  or  advanced  in  preg- 
nancy or  suffering  from  other  diseases  should  not  be  tested.  A 
positive  test  should  show  an  increase  of  at  least  1.5°  above  the 
previous  maximum  recorded  temperature.  The  temperature 
usually  begins  to  rise  in  about  eight  hours,  and  reaches  its  maximum 
in  from  ten  to  eighteen  hours  after  injection,  then  gradually 
subsides.  There  are  many  theories  of  the  mechanism  of  the  tuber- 
culin reaction,  but  it  is  now  believed  to  be  explained  best  on 
the  basis  of  anaphylaxis.  The  fact  that  the  amount  of  tuberculin 
used  in  the  test  is  not  appreciably  poisonous  to  a  healthy  animal 
indicates  that  the  infected  animal  has  become  sensitized  against 
the  bacterial  constituents,  probably  proteins  The  presence  of  a 
specific  allergin  has  not  been  satisfactorily  demonstrated,  but  some- 
thing of  that  nature  is  probably  present  and  renders  the  tissues 
sensitive,  principally  about  the  lesions.  That  this  sensitiveness 
extends  to  other  tissues  also  is  shown  by  the  ophthalmic  and  other 
reactions  to  be  described  presently.  It  is  a  well-known  fact  that 
one  injection  of  tuberculin  will  prevent  a  reaction  to  a  second 
injection  made  shortly  after.  This  fact  is  made  use  of  by  dishonest 
cattlemen  in  vitiating  the  tuberculin  test.  The  probable  explana- 
tion of  this  fact  is  that  the  body  is  in  a  condition  of  anti-anaphy- 
laxis,  that  the  allergin  has  been  exhausted  by  the  preceding  dose, 
and  there  has  been  insufficient  time  for  the  accumulation  of  a  new 
supply.  There  is  no  evidence  that  the  use  of  tuberculin  as  or- 
dinarily practised  ever  results  in  the  sensitization  of  the  animal. 
This  is  an  important  point,  as  the  test  can  be  used  repeatedly  in  a 
herd  of  cattle,  and  the  results  may  be  relied  upon.  Animals  in 
advanced  stages  of  the  disease  frequently  fail  to  react.  In  such 
cases  it  is  probable  that  the  body  is  in  a  state  of  immunity  to  the 
proteins  of  the  tubercle  bacillus.  As  was  stated  in  the  discussion 
of  anaphylaxis,  the  mechanism  of  this  immunity  is  not  well  under- 
stood. This  immunity  to  injections  of  tuberculin  must  not  be 
confused  with  immunity  to  the  disease,  for  it  seems  that  these 


ACID-FAST   GROUP  323 

rest  upon  quite  different  factors.  The  diagnosis  of  tuberculosis 
in  the  human  is  generally  accomplished  by  other  means  than  in- 
jection, on  account  of  the  severity  of  the  reactions.  In  cattle  it 
may  be  relied  upon  quite  implicitly  if  the  test  is  carried  out  prop- 
erly. 

Von  Pirquet  has  described  a  cutaneous  tuberculin  reaction  that 
is  of  diagnostic  value  in  man,  particularly  in  young  children. 
The  iimer  side  of  the  forearm  is  washed  with  ether.  One  drop 
of  old  tuberculin  is  applied,  and  another  at  a  distance  of  about 
10  cm.  The  skin  is  slightly  scarified  and  the  drops  rubbed  with  a 
bit  of  cotton.  A  positive  diagnosis  is  evidenced  by  the  develop- 
ment of  a  papule  resembling  that  of  vaccinia.  The  reaction  is 
quite  local,  showing  that  the  tissues  of  the  body  distant  from  the 
tuberculous  lesions  have  been  sensitized.  The  method  has  been 
extensively  tested  and  has  been  found  quite  accurate.  Von 
Pirquet  secured  positive  reactions  in  87  per  cent,  of  the  clinically 
tubercular,  in  20  per  cent,  of  clinically  tubercular  free.  The 
reaction  was  found  to  be  far  more  accurate  during  the  first  year 
of  life  than  later.  Lignieres  modified  this  method  by  shaving  the 
skin  and  avoiding  scarification.  Six  drops  of  undiluted  old  tuber- 
culin are  applied  and  rubbed  with  a  bit  of  absorbent  cotton.  The 
reaction  is  similar  to  the  preceding.  It  has  been  termed  the  anti- 
tuberculin  reaction.  The  intradermal  method  has  yielded  satis- 
factory results  when  used  upon  cattle  by  some  investigators;  others 
find  it  less  reliable  than  subcutaneous  injection. 

The  ophthalmo-reaction  of  Calmette  and  the  conjunctive^,  reaction 
of  Wolff-Eisner  have  likewise  found  extensive  use  in  recent  years 
in  human  diagnosis.  A  tuberculin  specially  prepared  free  from 
gtycerin  and  other  irritants  is  dropped  into  the  conjunctival 
sac.  A  positive  reaction  consists  in  the  appearance  of  a  pronounced 
conjunctivitis,  which  shows  itself  in  five  or  six  hours  and  disappears 
in  two  or  three  days.  This  reaction  may  be  used  upon  cattle,  but 
it  is  not  as  specific  as  the  subcutaneous  injection. 

Transmission  and  Prophylaxis. — Channels  Through  Which 
the  Organisms  Leave  the  Body. — These  are  determined  largely  in 
both  man  and  animals  by  the  localization  of  the  lesions.  The 
organism  is  to  be  found  in  the  sputum  in  man  and  animals  affected 
by  pulmonary  tuberculosis.  The  infectious  droplets  thrown  out 


324  VETERINARY   BACTERIOLOGY 

in  coughing  and  the  contamination  of  drinking- vessels  are  common 
sources  of  infection.  Material  coughed  up  from  the  lungs  by 
cattle  is  commonly  swallowed,  and  both  pulmonary  and  intestinal 
tuberculosis  in  these  animals  results  in  large  numbers  of  organ- 
isms being  thrown  off  with  the  feces.  This  is  probably  the  most 
important  channel  of  exit  in  the  cow.  Tuberculosis  in  swine  fol- 
lowing cattle  is  undoubtedly  due  to  the  ingestion  of  bacteria  voided 
in  this  manner.  Contamination  of  milk  with  tubercle  bacilli  is 
almost  inevitable  when  they  are  constantly  present  in  the  feces. 
Milk  is  also  found  to  contain  tubercle  bacilli  when  lesions  are 
present  in  the  udder.  Whether  or  not  they  may  be  present  when 
the  udder  is  not  tuberculous  is  a  mooted  question,  but  as  the 
bacilli  can  rarely  if  ever  be  demonstrated  in  the  blood,  it  is  not 
probable  that  they  can  enter  the  milk  direct  when 'the  udder  lesions 
are  absent.  The  urine  may  occasionally  contain  the  bacteria. 

The  disease  is  probably  never  inherited,  the  offspring  being  in- 
fected only  when  the  disease  infects  the  uterus  and  the  placenta. 
This  fact  is  of  importance,  for  upon  it  the  Danish  veterinarian, 
Bang,  has  outlined  a  practicable  method  of  building  up  herds  free 
from  tuberculosis.  The  calves  are  separated  from  their  tuberculous 
mothers  soon  after  birth  and  are  fed  only  upon  pasteurized  milk. 
Those  animals  known  to  be  tuberculous  are  separated  from  the 
others  and  a  quarantine  strict  enough  to  prevent  the  transfer  of 
the  disease  from  infected  to  non-infected  animals  is  established. 

Infection  Atria  in  Tuberculosis. — The  portals  of  entry  of 
Bacillus  tuberculosis  have  been  investigated  at  length  in  recent 
years,  but  there  are  still  discrepancies  in  the  results  of  investigators 
that  are  unexplained.  One  group  of  tuberculous  infections,  par- 
ticularly the  pulmonary  type  in  man,  is  probably  due  to  inhalation 
of  the  organisms.  This  was  at  one  time  universally  conceded,  but 
the  work  of  Calmette  and  others  has  shown  that  primary  pul- 
monary tuberculosis  may  result  from  ingestion  of  the  organisms. 
Certain  investigators  believe  this  to  be  far  more  common  than  in- 
fection by  inhalation.  It  has  been  shown  that  the  tubercle  bacilli 
may  be  demonstrated  in  the  thoracic  duct  of  a  dog  fed  upon  the 
organisms  within  a  few  hours  after  their  ingestion,  and  without 
any  apparent  lesion  of  the  intestinal  wall.  The  alimentary  tract 
is  undoubtedly  the  infection  atrium  in  most  cases  of  intestinal 


ACID-FAST   GROUP  325 

tuberculosis  and  of  infection  of  the  other  abdominal  organs,  the 
mesentery,  and  the  peritoneum.  The  tonsils  are  believed  with 
good  reason  to  permit  the  infection  of  the  neighboring  lymph- 
glands  and  the  consequent  production  of  scrofula.  Cutaneous 
lesions  following  abrasions  or  cuts  of  the  skin  have  been  observed, 
particularly  in  butchers,  and  in  a  few  instances  in  veterinarians. 

Intertransmissibility  of  Human,  Bovine,  and  Avian  Tuberculosis. 
— Human  tubercle  bacilli  do  not  readily  infect  cattle.  When 
injected,  they  may  cause  local,  but  rarely  general,  infection.  The 
fact  that  the  human  and  bovine  types  of  bacilli  may  be  differen- 
tiated has  already  been  emphasized.  Both  types  of  bacilli  produce 
fatal  infection  in  the  monkey.  Instances  of  probable  cutaneous 
infections  of  man  from  cattle  are  on  record. 

Park  and  Krumweide  have  outlined  the  factors  which  must 
be  taken  into  consideration  for  differentiation  of  the  bovine  and 
human  type  of  tubercle  bacilli.  In  their  own  isolations  of  the 
tubercle  bacilli  from  the  human  they  have  used  the  following 
criteria:  "  All  cultures  showing  maximum  luxuriance  on  glycerin 
egg  in  primary  cultures  are  certainly  human.  All  cultures  not 
showing  maximum  luxuriance  should  then  be  transferred  to 
glycerin  egg,  potato,  and  glycerin  egg  with  bouillon  added.  Cul- 
tures which  have  failed  on  glycerin  egg  should  also  be  transferred 
on  plain  egg,  as  glycerin  egg  may  again  fail.  All  cultures  showing 
maximum  luxuriance  are  quite  certainly  human."  The  forms 
which  show  a  very  sparse  growth  are  as  quite  certainly  bovine. 
Intermediate  forms  are  tested  upon  rabbits,  and,  if  necessary,  upon 
calves. 

Swine  readily  contract  bovine  tuberculosis.  They  have  also 
been  known  to  become  infected  with  avian  tuberculosis  through 
the  consumption  of  birds  dead  from  the  disease. 

Birds  contract  the  disease  from  each  other  by  ingestion  of 
excreted  bacteria.  Whether  or  not  the  disease  may  start  from 
ingestion  of  bovine  or  human  tubercle  bacilli  is  not  certainly 
known. 

It  is  evident  that  while  each  of  the  types  of  tubercle  bacilli  are 
generally  associated  with  a  specific  host,  on  the  other  hand, 
each  may,  under  appropriate  conditions  of  infection  and  suscep- 
tibility, produce  infection  in  other  hosts. 


326 


VETERINARY    BACTERIOLOGY 


The  following  table,  adapted  from  a  paper  by  Park  and  Krum- 
weide,  summarizes  1042  cases  of  human  tuberculosis  reported  in 
which  the  bovine  or  human  character  of  the  organism  was  deter- 
mined. The  age  groupings  are  particularly  interesting. 


DIAGNOSIS. 

ADULTS 
16  yrs.  and  over. 

CHILDREN 
5-16  yra. 

CHILDREN 
Under  5  yrs. 

Human 

Bovine 

Human 

Bovine 

Human 

Bovine 

Pulmonary  tuberculosis  
Axillary      inguinal      tubercular 
adenitis  
Cervical  tubercular  adenitis 

568 

2 
22 

15 

6 

28 

4 

K?) 

1 
3 

1 

11 

4 
33 

7 

2 
4 

1 

7 
2 
26 
2 

20 

7 

3 
1 

'  1 

1 

12 

2 
15 

6 

13 

28 

3 

45 
14 
21 
2 

20 
13 

10 

5 

8 

1 
2 

Abdominal  tuberculosis 

Generalized    tuberculosis  of  ali- 
mentary origin                   

Generalized  tuberculosis  
Generalized     tuberculosis     and 
meningitis  of  alimentary  origin 
Generalized     tuberculosis     and 
meningitis 

Meningitis 

Tuberculosis  of  bonas  and  joints  . 
Other  types                                  .  . 

18 
14 

1 
2 

Totals  

677 

9 

99 

33 

161 

59 

There  seems  to  be  no  reasonable  doubt  but  that  the  bovine 
tubercle  bacillus  may  infect  the  human.  The  above  table  shows 
it  to  be  common  enough  in  children  under  sixteen  years  to  justify 
all  reasonable  precautions  against  the  ingestion  of  infected  meat 
and  milk.  The  danger  to  the  adult  would  appear  to  be  almost 
negligible. 

Bacillus  of  Johnes'  Disease 

Disease  Produced. — Chronic  enteritis  or  para-tubercular  dys- 
entery of  cattle. 

Johnes  and  Frothingham,  in  1895,  described  an  acid-fast 
organism  as  the  probable  cause  of  chronic  dysentery  in  cattle. 
They  believed  the  organism  to  be  identical  with  the  avian  tubercle 
bacillus,  but  this  has  been  disproved  by  subsequent  investigations. 
It  has  not  received  a  specific  name. 

Distribution. — The  disease  has  been  noted  in  various  parts  of 
Europe  and  in  the  United  States.  Cases  have  been  reported  from 
Wisconsin,  Minnesota,  and  Iowa. 


ACID-FAST   GROUP  327 

Morphology  and  Staining. — The  organism  closely  resembles  the 
Bacillus  tuberculosis  morphologically.  It  is  a  slender  rod,  usually 
1  to  2  /^  in  length,  rarely  longer.  It  is  non-motile.  Neither 
spores  nor  capsules  have  been  observed.  It  does  not  stain  readily 
with  the  aqueous  anilin  dyes  unless  mordanted.  When  once 
stained,  it  is  acid-fast.  The  same  staining  technic  may  be  used 
as  with  the  tubercle  bacillus. 

Isolation  and  Culture. — Bugge  and  Albein  claim  to  have  isolated 
the  organisms,  but  details  are  lacking.  It  has  not  been  culti- 
vated by  other  investigators. 

Pathogenesis. — Experimental  Evidence. — The  disease  has  not 
been  transmitted  to  any  of  the  laboratory  experimental  animals. 
The  belief  that  it  is  the  etiologic  factor 

in  the  disease  rests  upon  its  constant         *:V^?-:^-'^^  •&<;-.- 
presence  in  the  lesions.  €^:^  ^   ~i 

Character  of  Disease  and  Lesions  Pro-  :^'^--</^-^:-  ^ 
duced. — The  disease  is  characterized  by  V- '•'/$•  ]j.9l'j$? t%^' 
progressive  emaciation  and  persistent  -^i^^^>^^ 
chronic  diarrhea,  with  commonly  fatal  "C'/^^^^v.®::'''--s| 

termination.     The  lesions  are  confined  »>  :      3S& 

*%£>%  ©    ' '  A '-'    •  '  4ft 
to  the  intestines,  small  intestines  pri- 

.,  ,  ,,          ,  T     .,         mv  Fig.     132. — Bacillus     of 

manly,  and  the  colon  secondarily.     The      T  ,      ,   ,. 

tj  oniics    QisG3.sc  iri  section 

mucosa  shows  thickening  and  wrinkling,      Of  intestinal  wall, 
but  there  is  little  evidence  of   conges- 
tion.    The  lesions  are  not  sharply  limited,  there  is  no  necrosis, 
although  some  of  the  villi  may  be  denuded  of  epithelium.     Giant- 
cells  may  rarely  be  demonstrated,  but  tubercle  formation  does  not 
occur. 

Immunity. — Nothing  is  known  relative  to  immunity  to  this 
disease. 

Bacteriologic  Diagnosis. — No  practicable  method  of  bac- 
teriological antemortem  diagnosis  has  been  developed.  The 
presence  of  acid-fast  organisms  in  large  numbers  in  the  thickened 
mucosa  in  the  absence  of  nodule  formation  is  diagnostic  on  post- 
mortem. 

Transmission. — The  disease  is  doubtless  transmitted  by  inges- 
tion,  but  this  has  not  been  experimentally  demonstrated. 


328  VETERINARY    BACTERIOLOGY 

Bacillus  leprae 

Synonyms. — Bacterium  leprce;  Mycobacterium  leprce. 

Disease  Produced. — Leprosy  in  man. 

Hansen,  in  1872,  discovered  the  bacillus  of  leprosy  in  the  lesions 
of  the  disease.  It  was  studied  more  at  length  by  Neisser  and 
Hansen,  who  published  their  report  in  1880. 

Distribution. — Leprosy  is  common  in  Asia,  northern  Europe, 
in  certain  Pacific  Islands,  and  is  found  occasionally  in  various 
parts  of  the  United  States.  A  similar,  though  probably  not 
identical,  disease  has  been  noted  by  Wherry  and  others  in  rats. 

Morphology  and  Staining. — B.  kprce  resembles  the  B.  tubercu- 
losis. It  is  a  slender  rod,  frequently  as  much  as  6  ^  in  length.  The 


Fig.  133. — Bacillus  leprce  (Kolle  and  Wasserman). 

rods  are  usually  straight,  non-motile,  do  not  produce  spores  or 
capsules.  They  stain  somewhat  more  readily  than  the  B.  tuber- 
culosis, but  are  distinctly  acid-fast. 

Isolation  and  Culture. — Several  investigators  claim  to  have 
cultivated  the  leprosy  bacillus,  but  the  results  in  every  (jase  need 
confirmation.  It  is  not  certainly  known  that  it  has  ever  been  suc- 
cessfully grown  upon  artificial  media. 

Physiology. — Until  the  organism  can  be  secured  in  pure  cul- 
tures, little  or  nothing  can  be  determined  as  to  its  physiology. 

Pathogenesis. — Experimental  Evidence. — Lower  animals,  with 
the  possible  exception  of  the  monkey,  cannot  be  successfully 
infected  with  the  Bacillus  leprce.  Arning  succeeded  in  infecting 


ACID-FAST   GROUP  329 

a  criminal  in  the  Hawaian  Islands  by  subcutaneous  implantation 
of  leprous  tissue.  The  belief  in  the  pathogenicity  of  this  organism 
rests  principally  upon  its  presence  in  great  numbers  in  the.  leprous 
tissues. 

Character  of  Disease  and  Lesions  Produced. — The  organisms  may 
proliferate  in  the  nerves,  causing  anesthetic  leprosy;  or  in  the 
subcutaneous  tissues,  producing  nodules  resembling  superficially 
those  of  tuberculosis.  The  disease  progresses  slowly,  the  infected 
individual  surviving  for  years  or  even  several  decades. 

Immunity. — Methods  of  immunization  are  unknown. 

Bacteriologic  Diagnosis. — The  disease  may  be  diagnosed  bac- 
teriologically  by  scraping  a  nodule  and  preparing  a  smear  from 
the  serum  so  obtained.  The  characteristic  acid-fast  organism 
may  be  demonstrated.  In  the  hands  of  some  investigators  the 
complement  binding  reaction  (using  extracts  from  leprous  organs 
as  the  antigen)  has  proved  of  diagnostic  value. 

Transmission. — The  method  of  the  spread  of  leprosy  is  not 
understood. 

Non-pathogenic  Acid-fast  Bacteria 

Many  species  of  non-pathogenic  acid-fast  bacteria  have  been 
described,  and  from  a  variety  of  sources.  Some  are  normal 
inhabitants  of  the  skin,  others  occur  in  dung  and  in  soil.  The 
presence  of  these  organisms  upon  the  body,  in  milk,  etc.,  frequently 
renders  a  differential  diagnosis  necessary  between  them  and  the 
Bacillus  tuberculosis.  A  few  of  the  more  important  types  will  be 
briefly  described. 

Bacillus  smegmatis. — This  organism  has  been  repeatedly 
observed  in  the  smegma  from  the  genitals  of  man  and  animals 
and  from  the  skin  of  the  axillae.  Morphologically  it  resembles 
the  tubercle  bacillus.  Its  isolation  is  accomplished  with  consider- 
able difficulty  and  only  upon  media  containing  blood-serum.  The 
B.  smegmatis  is  non-pathogenic.  It  is  of  importance  principally 
because  of  the  possibility  of  confusion  with  B.  tuberculosis  when 
it  occurs  in  the  urine.  Although  the  smegma  bacillus  is  acid  fast, 
it  may  be  decolorized  by  alcohol  according  to  most  authors. 

Dung  Bacillus,  Grass  Bacillus  of  Moeller,  Butter  Bacillus,  etc. 
— Acid-fast  bacteria  not  unlike  the  B.  tuberculosis  in  morphology 
have  been  isolated  from  a  great  variety  of  sources.  They  may  in 


330 


VETERINARY   BACTERIOLOGY 


general  be  easily  differentiated  from  the  tubercle  bacillus,  as  they 
do  not  produce  a  generalized  tuberculosis  when  inoculated  into 
experimental  animals.  However,  nodules  resembling  those  of 
tuberculosis  are  found  as  a  result  of  the  injection  of  some  species. 
The  organisms  when  isolated  upon  culture-media,  however,  are 
found  to  develop  luxuriantly  even  at  room-temperatures.  It  is 


Fig.  134. — Bacillus  smegmatis,  in  a  stained  smear  of  preputial  smegma  (Frankel 

and  Pfeiffer). 

interesting  to  note  that  isolation  of  such  bacteria  from  soil  has  been 
accomplished  by  the  use  of  antiformin.  The  differential  diagnosis 
of  these  forms  from  B.  tuberculosis  must  always  be  accomplished 
by  both  animal  inoculation  and  isolation  upon  culture-media. 
In  making  a  diagnosis  of  tuberculosis  from  stained  mounts  the  pos- 
sible presence  of  these  forms  must  constantly  be  borne  in  mind. 


CHAPTER  XXXI 

ANTHRAX  GROUP 

THE  organisms  Bacillus  anthrads  and  Bacillus  lactimorbi  are 
included  in  this  group.  It  is  possible  that  the  latter  organism 
might  better  constitute  the  type  of  another  group,  but  it  resembles 
the  B.  anthrads  in  a  sufficient  number  of  its  morphological  charac- 
ters to  render  a  tentative  association  of  the  two  forms  justifiable. 

The  organisms  belonging  to  this  group  are  aerobic  bacilli  which 
form  spores,  are  gram-positive,  and  liquefy  gelatin.  These  two 
bacilli  are  the  only  pathogenic  aerobic  spore  producers  which  have 
been  described. 

This  group  is  in  reality  a  subdivision  of  the  much  larger  Badllus 
subtilis,  or  hay-bacillus  group  of  bacteria.  Organisms  closely 
related  morphologically  and  culturally  to  the  Badllus  anthrads 
are  ubiquitous ;  they  are  found  by  tens  of  thousands  in  every  gram 
of  most  surface  soils.  A  dozen  or  more  species  of  these  soil  organ- 
isms have  been  described,  some  of  them  so  similar  to  the  anthrax 
bacillus  that  they  can  practically  be  differentiated  only  by  suitable 
animal  inoculations.  These  bacteria  are  among  the  most  active 
in  nature  in  bringing  about  the  decomposition  of  organic  substances 
in  the  soil,  particularly  in  the  series  of  changes  grouped  together 
under  the  heading  of  Ammonincation. 

Bacillus  anthracis 

Synonym. — Bacterium  anthrads. 

Diseases  Produced. — Anthrax,  splenic  fever,  Milzbrand,  char- 
bon  in  cattle,  sheep,  and  rarely  other  animals;  malignant  car- 
buncle, woolsorters'  disease,  in  man. 

Pollender,  in  1849,  observed  B.  anthrads  as  minute  rods  in 
the  blood  of  cattle  which  had  died  of  anthrax.  Davaine  also 
observed  and  described  it  in  the  following  year.  The  complete 
proof  that  this  organism  causes  the  disease  was  furnished  by  the 

331 


332 


VETERINARY    BACTERIOLOGY 


work  of  Dr.  Robert  Koch,  published  in  1876.  Methods  of  vac- 
cination were  developed  by  Toussaint  in  1880,  and  by  Pasteur 
in  1881. 

Distribution. — Anthrax  has  been  recorded  in  Europe  since 
ancient  times.  It  is  known  also  from  Africa,  Asia,  Australia, 
and  South  America.  It  has  been  reported  from  about  one-third 
of  the  States  of  the  United  States,  and  probably  occurs  sporadically 
in  most  of  them.  The  disease  in  this  country  is  not  common,  ex- 
cept in  a  few  thoroughly  infected  localities. 

Morphology  and  Staining. — Bacillus  anthracis  is  a  large  rod, 
straight,  usually  with  truncate  ends,  1  to  1.25  by  4.5  to  10  /w,  in 
short  chains  when  examined  in  tissues  or  blood  and  in  long  chains 

in  culture-media.  It  is  non- 
motile.  Capsules  may  be 
demonstrated  in  smears  from 
blood  and  other  tissues. 
Spores  are  produced  only 
when  the  organism  is  grown 
in  the  presence  of  free  oxygen. 
They  do  not,  therefore,  occur 
in  the  bacillus  as  found  in  the 
blood  and  the  tissues.  The 
single  spore  produced  by  a 
cell  is  oval  or  spherical,  oc- 
cupies the  middle  of  the  cell, 
and  is  of  almost  the  same 
diameter.  The  spores  are 

much  more  refractive  than  the  protoplasm  of  the  non-sporulating 
cells.  The  spores  germinate  on  being  brought  under  favorable 
growth  conditions  by  breaking  through  the  spore-wall  at  the  pole. 
They  may  be  demonstrated  by  a  contrast  spore  stain.  The 
vegetative  rod  stains  readily  with  the  aqueous  anilin  dyes  and 
is  gram-positive.  Metachromatic  granules  may  rarely  be  demon- 
strated. 

Isolation  and  Culture. — B.  anthracis  may  be  readily  isolated 
in  pure  culture  upon  any  of  the  common  laboratory  media  by 
direct  inoculation  from  the  blood  of  an  infected  animal  or  from 
the  internal  organs,  particularly  the  spleen  or  liver.  Plate  cul- 


Fig.  135. — Bacillus  anthracis,  rods  with- 
out spores  (Giinther). 


ANTHRAX   GROUP  333 

tores  in  agar  or  gelatin  are  sometimes  necessary  when  the  organism 
is  mixed  with  other  forms.  The  colonies  microscopically  are 
found  to  consist  of  long  chains  of  bacilli,  which,  under  the  low 
power,  resemble  tufts  of  curled  hair.  This  appearance  is  quite 
characteristic,  but  is  closely  duplicated  by  certain  soil  organisms 
of  the  Bacillus  subtilis  group.  In  gelatin  stabs  a  "  spiking " 
occurs,  i.  e.,  filaments  radiate  from  the  line  of  puncture  and  give 
the  appearance  of  an  inverted  fir  tree.  The  gelatin  is  liquefied 
slowly.  The  growth  on  potato  is  creamy  in  color  and  rather  dry 
in  consistency.  Blood-serum  is  slowly  liquefied.  Milk  is  rendered 
slightly  acid,  curdled  by  a  lab  ferment,  and  the  casein  digested. 


, 


'- 

Fig.  136.  —  Bacillus  anthracis,  with  spores  (Frankel  and  Pfeiffer). 

In  bouillon  the  organism  frequently  forms  a  pellicle  which  readily 
settles  to  the  bottom.  Clouding  of  the  medium  does  not 
occur. 

Physiology.  —  Bacillus  anthracis  grows  best  in  the  presence  of 
oxygen.  The  optimum  growth  temperature  is  about  37°.  It 
will  grow  at  temperatures  as  high  as  45°,  and  also  at  room-temper- 
ature. The  vegetative  rods  are  easily  destroyed  by  heat,  but  the 
spores  must  be  heated  to  100°  for  five  minutes  before  they  are 
certainly  killed.  The  spores  likewise  exhibit  great  resistance 
to  desiccation.  Dried  upon  threads,  they  have  been  known  to 
retain  their  vitality  for  years.  Five  per  cent,  phenol  destroys 


334  VETERINARY   BACTERIOLOGY 

the  spores  only  after  prolonged  contact.  A  rennet-like  enzyme 
and  ferments  which  digest  casein,  gelatin,  and  blood-serum  are 
produced.  Acids  and  gas  are  not  formed. 

Pathogenesis. — Experimental  Evidence. — Guinea-pigs,  mice,  and 
rabbits  are  very  susceptible  to  experimental  inoculation.  The 
subcutaneous  injection  of  anthrax  bacilli  results  in  the  develop- 
ment of  a  quickly  fatal  septicemia  having  the  characters  of  the 
disease  as  it  occurs  in  the  larger  animals.  The  carnivora  are 
relatively  immune.  Birds  show  a  high  degree  of  immunity. 
Inoculation  of  cattle,  sheep,  swine,  and  other  susceptible  domestic 
animals  with  pure  cultures  of  the  organism  invariably  produces 


Fig.  137. — Bacillus  anthrads  colony       Fig.    138. — Bacillm    anthracis,   stab 
(Giinther).  culture  in  gelatin  (Giinther). 

the  disease,  so  that  there  is  no  question  of  the  causal  relationship 
of  this  organism. 

Character  of  Disease  and  Lesions  Produced. — As  it  occurs  in 
animals  the  disease  is  usually  a  distinct  and  rapidly  fatal  septi- 
cemia. Hemorrhagic  and  serous  infiltration  of  the  subcutaneous 
and  other  connective  tissues  and  subepidermal  hemorrhages  are 
characteristic.  The  spleen  is  much  enlarged,  often  several  times 
its  normal  size.  The  liver,  kidneys,  and  lungs  are  usually  congested 
and  ecchymotic.  The  organism  is  to  be  found  in  great  numbers 
everywhere  in  the  blood-stream.  Cutaneous  infections  in  horso 
and  man,  rarely  in  cattle,  develop  as  carbuncles  from  which  the 
organism  may  not  invade  the  general  circulation,  and  healing 
may  occur.  Commonly,  however,  this  infection  terminates 


ANTHRAX  GROUP 


335 


fatally.       Pulmonary    infection   resulting   in  a  pneumonia  with 
quickly  fatal  termination  may  occur  from  inhalation. 

Immunity. — No  true  toxin  has  been  demonstrated  for  the 
anthrax  bacillus,  in  fact,  it  is  difficult  to  account  for  the  patho- 
genicity  of  the  organism  on  the  basis  of  specific  poison  formation. 
Immune  serum  is  claimed  by  some  investigators  to  have  a  con- 
siderable specific  agglutinative  power,  but  others  have  failed  to 
demonstrate  this.  It  seems  that  this  reaction  is  inconstant  and 
may  be  entirely  absent,  even  though  the  serum  have  a  high  im- 
munizing value.  Certain  normal  bloods,  as  from  the  dog,  show 
bacteriolytic  power,  but  specific  bacteriolysins  cannot  be  demon- 
strated in  most  immune  sera.  Opsonins,  both  normal  and  im- 


Fig.  139. — Bacillus  anthracis,  stained  mount  of  blood,  showing  the  capsules  of 
the  bacilli   (Preisz). 

mune,  have  been  demonstrated.  Aggressins,  according  to  Bail, 
account  for  its  pathogenicity.  Many  theories  of  immunity  in 
anthrax  have  been  developed,  but  none  of  fhem  has  been  gener- 
ally accepted  as  solving  the  problem  wholly. 

Active  immunization  by  vaccination  has  been  extensively 
practised.  The  organism  may  be  attenuated  in  a  variety  of  ways. 
The  first  developed  was  that  of  Pasteur,  and  it  is  still  the  one  most 
commonly  used.  The  organism  is  grown  at  a  temperature  of 
42°  to  43°  for  varying  lengths  of  time.  The  pathogenicity  grad- 
ually decreases  until  injections  no  longer  kill  the  rabbit;  longer 
growth  attenuates  it  until  the  guinea-pig  is  not  susceptible,  and 
finally  even  the  mouse  will  not  succumb.  The  exact  length  of 
time  required  for  attenuation  in  each  instance  can  be  determined 


336  VETERINARY   BACTERIOLOGY 

only  by  experimentation,  as  there  are  many  unknown  or  uncon- 
trolled factors  involved.  Chamberland  and  Roux  found  that  atten- 
uation could  likewise  be  accomplished  by  the  use  of  small  amounts 
of  certain  antiseptics  in  the  culture-medium.  One  part  in  600  : 
800  of  phenol,  and  one  part  in  2000  :  5000  of  potassium  bichro- 
mate were  found  to  reduce  the  virulence  so  that  ten  days'  exposure 
rendered  the  organism  non- virulent  for  the  sheep.  Numerous  other 
methods  of  attenuation  have  been  proposed,  such  as  heating  to  a 
temperature  just  below  the  thermal  death-point,  and  injection  into 
resistant  animals,  such  as  the  frog  and  white  rat.  These  latter  have 
no  practical  significance.  Before  use  the  virulence  of  cultures  grown 
from  sixteen  to  eighteen  hours  at  37°  must  be  determined.  Rab- 
bits, guinea-pigs,  and  mice  are  inoculated  with  varying  amounts — 
usually  from  y1^  to  unnr  °f  a  standard  loopful  of  the  culture  is  in- 
jected subcutaneously.  An  exposure  to  a  temperature  of  42.5°  for 
eleven  days  renders  the  organism  non- virulent  for  the  rabbit,  but 
an  exposure  of  twenty  days  is  required  before  it  shows  diminished 
virulence  for  the  guinea-pig,  and  of  ten  weeks  before  it  becomes 
non-virulent  for  the  mouse.  Experiments  have  shown  that  the 
organisms  once  attenuated  do  not  regain  at  once  their  virulence 
when  cultivated  at  37°.  Pasteur  believed  that  the  attenuated 
forms  lost  their  power  of  spore  production,  but  avirulent  types 
have  since  been  shown  to  be  sporogenous  and  not  to  be  differen- 
tiated morphologically  from  the  fully  virulent  form.  Slight 
cultural  differences  have  been  described.  Pasteur  prepared  vac- 
cine of  two  grades  of  virulence.  The  "  Premier  vaccine  "  (vaccine 
I.)  killed  white  mice  with  certainty,  was  irregular  in  pathogenicity 
for  guinea-pigs  and  a1  virulent  for  rabbits.  The  "  Deuxieme  vac- 
cine "  (vaccine  II.)  killed  guinea-pigs  and  was  somewhat  virulent 
for  the  rabbit.  About  0.25  c.c.  of  vaccine  I.  is  used  for  cattle,  half 
the  amount  for  sheep.  Twelve  days  later  a  similar  injection  of  vac- 
cine II.  is  made.  The  use  of  the  Pasteur  vaccination  method  has 
been  attended  with  highly  satisfactory  results  in  European  coun- 
tries, although  not  so  good  in  the  United  States.  Variations  in  the 
virulence  of  vaccine  II.  and  also  differences  in  susceptibility  in 
the  vaccinated  animals  lead  to  a  loss  among  such  animals  which 
averages  about  1  per  cent,  in  cattle  and  somewhat  more  in  sheep  us 
a  direct  result  of  the  vaccination.  Many  modifications  of  the 


ANTHRAX    GROUP  337 

original  Pasteur  method  have  been  suggested,  but  in  only  slightly 
modified  form  it  is  the  one  still  most  commonly  used. 

Attempts  to  used  killed  cultures  of  anthrax  bacilli  or  their 
sterile  products  in  producing  immunity  have  failed  to  yield  satis- 
factory results  in  practice.  Bail  claims  that  an  active  immunity 
may  be  established  by  the  use  of  aggressins.  The  material  used  is 
the  serous  fluid  from  animals  dead  from  anthrax;  this  is  sterilized 
by  phenol  and  injected  into  an  animal  to  be  immunized.  The 
aggressin  should  preferably  be  secured  from  the  same  species  of 
animal  as  the  one  to  be  immunized.  The  immunity  is  not  estab- 
lished until  after  the  lapse  of  ten  days  or  more.  When  established, 
it  is  claimed  that  the  animal  will  resist  infection  with  fully  virulent 
cultures.  This  method  of  immunization  with  aggressins  has  not 
been  thoroughly  tested  out. 

Passive  immunization  by  means  of  antisera  has  been  tried  by 
numerous  investigators.  Cattle,  the  horse,  ass,  and  sheep  have 
been  used  in  the  production  of  such  sera.  The  animals  are  first 
immunized  by  Pasteur's  method  of  vaccination,  and  in  ten  days 
or  two  weeks  from  TWIT  t°  TO"  °f  a  1°°P  °f  a  fully  virulent  culture 
is  injected.  This  is  generally  followed  by  a  strong  reaction.  Two 
or  three  weeks  later  a  somewhat  larger  dose  is  given,  and  the  dosage 
is  gradually  increased  until  many  loopfuls — then  entire  cultures — 
are  injected.  In  three  to  four  months  an  immunity  is  developed 
such  that  the  subcutaneous  injection  of  several  cultures  from  agar 
slants  may  be  made  without  noteworthy  reactions.  The  blood 
is  drawn  about  two  weeks  after  the  last  injection.  The  animal 
may -then,  after  the  lapse  of  two  weeks,  be  again  injected  and  again 
bled.  The  serum  is  pipetted  off  and  preserved  with  0.5  per  cent, 
phenol.  Exact  methods  of  standardization  such  as  are  used  with 
antitoxins  cannot  be  employed.  An  approximation  of  the  potency 
can  be  reached  by  the  intravenous  injections  of  varying  amounts 
of  serum  into  rabbits,  and  five  to  ten  minutes  later  inoculating  each 
subcutaneously  with  y-^Vir  of  a  loop  of  a  virulent  culture.  A 
serum  is  regarded  as  usable  if  two  out  of  five  animals  injected  with 
2,  3,  4,  5,  and  6  c.c.  of  the  serum  survive,  and  the  remainder  live 
longer  than  control  animals.  As  noted  above,  the  immune  serum 
contains  agglutinins,  some  bacteriolysins,  and  opsonins  specific 
for  the  anthrax  bacilli.  The  serum  may  be  used,  both  in  prophy- 
22 


338  VETERINARY   BACTERIOLOGY 

laxis  and  cure.  It  is  of  therapeutic  value  in  man,  also.  Ten  c.c. 
is  a  prophylactic  dose  for  sheep.  The  serum  is  of  greatest  use 
where  it  is  necessary  to  immunize  large  numbers  of  animals  quickly, 
as  in  a  herd  of  sheep  in  which  anthrax  has  made  its  appearance. 
The  serum  has  not  been  used  extensively  enough  to  warrant  defi- 
nite statements  of  its  value,  although  many  favorable  reports  Save 
been  recorded.  > 

Simultaneous  injection  of  antisera  and  virulent  cultures  has 
been  advocated  by  Sobernheim.  Its  value  has  not  been  satis- 
factorily demonstrated. 

Bacteriologic  Diagnosis. — Smears  from  the  blood  or  tissues  of 
an  animal  show  short  chains  of  large,  gram-positive  rods,  with 
blunt  ends.  Capsules  may  be  demonstrated  in  the  blood.  A  stab 
culture  in  gelatin  will  give  the  characteristic  spiking.  Animal 
inoculations  may  be  used  where  the  organism  is  not  in  pure  culture. 

Transmission. — Anthrax  is  usually  transmitted  from  one  ani- 
mal to  another  by  ingestion,  more  rarely  through  skin  lesions. 
Cutaneous  infection  and  infection  by  inhalation  are  most  common 
in  man.  The  organism  does  not  sporulate  within  the  body. 
Dead  animals  should  be  burned  or  buried  deeply.  The  excretions 
from  an  infected  animal,  the  feces  in  particular,  contain  many 
bacteria  which  can  form  spores  on  leaving  the  body.  Pastures 
once  infected  may  remain  so  for  many  years,  as  the  spores  are  not 
readily  destroyed  by  desiccation  and  may  persist  in  the  soil  for  a 
long  time.  Blood-sucking  flies  sometimes  spread  the  disease  from 
one  animal  to  another  by  direct  inoculation.  Care  should  always 
be  used  in  dealing  with  infected  animals,  as  the  disease  is  fatal  to 
man  as  well. 

Bacillus  lactimorbi 

Diseases  Produced. — Trembles  of  cattle;  milk  sickness  of  man; 
"  alkali-poisoning  "  in  the  southwestern  United  States. 

Jordan  and  Harris,  in  1909,  described  the  Bacillus  lactiinurl>i 
as  the  probable  cause  of  trembles  in  cattle  and  of  milk  sickness 
in  man.  The  organism  was  first  isolated  in  an  outbreak  in  Xcw 
Mexico. 

Distribution. — The  disease  has  been  reported  from  Ohio, 
Tennessee,  Carolina,  Kentucky,  Illinois,  Indiana,  and  the  south- 
western states  and  territories. 


ANTHRAX   GROUP  339 

Morphology  and  Staining. — The  Bacillus  lactimorbi  is  a  rod 
somewhat  smaller  than  the  anthrax  bacillus,  usually  single  or  in 
pairs,  occasionally  in  filaments.  It  is  motile  by  means  of  10  to  15 
peritrichous  flagella.  Capsules  have  not  been  observed.  Spores 
are  produced  in  the  ends  of  the  rods,  and  are  slightly  greater  in 
diameter  than  the  rod  itself.  The  young  rods  stain  unevenly 
with  methylene-blue,  and  show  very  distinct  metachromatic 
granules.  The  organism  is  gram-positive. 

Isolation  and  Culture. — Jordan  and  Harris  isolated  the  organ- 
ism directly  upon  agar  plates  from  the  intestinal  contents — bile, 


Fig.  140. — Bacillus  lactimorbi,  showing  rods  and  spores  (X  1100)  (Jordan 
and  Harris  in  "  Journal  of  Infectious  Diseases"). 


spleen,  liver,  and  pericardial  fluid.  The  agar  colonies  are  small 
and  Streptococcus-like.  The  growth  on  agar  slants  is  moderate 
at  first,  later  more  luxuriant,  but  without  distinct  differential 
characters.  Gelatin  stabs  show  incipient  liquefaction  in  about 
ten  days.  Bouillon  is  somewhat  clouded,  and  a  well-defined 
pellicle  forms.  Milk  is  not  coagulated.  In  litmus  milk  the  reac- 
tion is  observed  to  become  more  alkaline,  and  there  may  be  some 
development  of  opalescence  due  to  the  great  increase  in  alkalinity. 
There  is  no  growth  upon  potatoes.  Blood-serum  is  not  liquefied. 

Physiology. — The  optimum  growth  temperature  is  probably 
about  37°,  although  good  development  takes  place  at  room-temper- 


340  VETERINARY  BACTERIOLOGY 

atures.  The  thermal  death-point  for  non-sporulating  rods  is 
55°  for  five  minutes,  and  for  the  sporulating  rods,  100°  for  fifteen 
minutes.  The  organism  is  aerobic.  Neither  gas  nor  acids  are 
produced  from  carbohydrates.  Gelatinase  is  produced,  but 
not  enzymes  that  will  proteolize  milk  or  blood-serum. 

Pathogenesis. — Experimental  Evidence. — The  proof  that  this 
organism  stands  in  an  etiologic  relation  to  the  disease  does  not 
seem  to  be  entirely  satisfactory.  An  organism  not  distinguishable 
from  this  has  been  isolated  from  soil,  dung,  and  hay  in  localities 
from  which  the  disease  has  not  been  reported.  Intraperitoneal 
injection  of  the  heart  blood  from  a  case  of  trembles  into  a  rabbit 
resulted  in  death,  and  the  organism  was  recovered  from  various 
internal  organs.  Subsequent  efforts  at  infection  with  pure  cul- 
tures failed.  Inoculations  into  the  guinea-pig  were  unsuccessful. 
Feeding  experiments  upon  dogs  and  cats  were  more  successful, 
and  a  disease  corresponding  to  milk  sickness  was  produced  when  the 
organisms  were  fed  in  large  quantities. 

Character  of  Disease  and  Lesions. — Jordan  and  Harris  give  the 
following  characterization  of  the  disease.  "  The  course  of  the 
disease  in  cattle  is  marked  by  lassitude  and  muscular  weakness, 
sometimes,  but  not  invariably,  accompanied  by  constipation. 
There  is  often  muscular  twitching  or  trembling,  and  occasionally 
signs  of  nervous  excitement.  In  man  there  is,  as  a  rule,  excessive 
vomiting,  and  obstinate  constipation  accompanied  by  great  weak- 
ness. The  temperature  is  normal  or  subnormal."  In  cattle,  the 
principal  lesion  observed  is  fatty  degeneration  of  the  liver.  Ecchy- 
moses  in  the  heart- wall,  the  liver,  and  spleen  have  been  noted. 

Immunity. — Experiments  relative  to  the  agglutinating  power  of 
serum  have  not  yielded  consistent  results.  Nothing  is  known  of 
methods  of  conferring  immunity. 

Bacteriologic  Diagnosis. — The  organism  may  be  isolated  in  pure 
culture  from  the  infected  organs  and  may  be  identified  by  its  char- 
acteristic morphology. 


CHAPTER  XXXII 

ABORTION  BACILLUS  GROUP 

OXE  organism  only,  the  Bacillus  abortus  of  Bang,  is  placed  in 
this  group.  It  should  be  noted  that  other  organisms  besides  the 
one  here  discussed  are  doubtless  occasionally  responsible  for  abor- 
tion in  cattle  and  other  animals. 

Bacillus  abortus 

Synonyms. — Abortion  bacillus  of  Bang;  Bacterium  abortum. 

Disease  Produced. — Contagious  abortion  in  the  cow. 

Bang,  in  1897,  described  a  Bacillus  as  the  probable  cause  of 
contagious  abortion  in  the  cow.  The  specific  organism  was  isolated 
with  difficulty.  It  has  been  isolated  since  that  time  in  Europe 
several  times,  and,  more  recently,  in  the  United  States.  Several 
investigators  in  the  United  States  have  described  members  of  the 
colon  group  and  of  other  groups  as  present  in  contagious  abortion, 
but  it  is  doubtful  whether  in  many  cases  appropriate  cultural 
methods  have  been  utilized  for  the  isolation  of  this  organism. 

Distribution. — Contagious  abortion  has  been  reported  from 
many  localities  on  the  continent  of  Europe  and  in  Great  Britain. 
It  is  known  to  occur  in  all  sections  of  the  United  States. 

Morphology  and  Staining. — Bacillus  abortus  is  very  small,  and, 
according  to  Nowak,  resembles  the  bacillus  of  chicken  cholera. 
Nowak,  on  the  basis  of  its  morphology,  groups  it  with  the  Pas- 
teurellas  or  hemorrhagic  septicemia  bacilli.  It  is  polymorphic* 
in  culture-media.  Involution  forms  occur  as  branched  and  clubbed 
types.  It  is  non-motile,  and  neither  capsules  nor  spores  have  been 
demonstrated.  It  stains  readily  by  the  aqueous  anilin  dyes, 
frequently  showing  polar  granules.  It  is  gram-negative. 

Isolation  and  Culture. — The  isolation  and  cultivation  of  Bacillus 
abortus  are  attended  with  peculiar  difficulties.  The  organism 
may  be  frequently  obtained  at  once  in  pure  culture  from  the 

341 


342  VETERINARY   BACTERIOLOGY 

heart  blood  or  the  intestines  of  an  aborted  fetus.  Bang  used  a 
medium  consisting  of  nutrient  agar,  to  which  he  added  liquid  gela- 
tin and  sterile  liquid  blood-serum.  These  tubes  were  inoculated 
with  suspected  material,  mixed  well,  and  kept  at  blood-heat. 
In  the  course  of  three  days  numerous  colonies  developed  in  a 
definite  stratum  a  few  millimeters  below  the  surface.  As  will  be 
noted  under  the  discussion  of  physiology,  the  organism  has  an 
unusual  relationship  to  oxygen,  and  the  amount  of  oxygen  needed 
for  its  development  is  to  be  found  at  the  depth  at  which  the  colo- 
nies form.  These  colonies  are  compact,  rounded,  or  somewhat 
irregular,  sometimes  showing  a  dense  nucleus  surrounded  by  a 


Fig.  141.  —  Bacillus  abortus  (Nowak). 

lighter  zone.  When  the  organism  is  present  in  impure  culture, 
as  in  the  vagina  of  the  cow,  other  methods  are  necessary  for  its 
isolation.  Nowak  has  described  a  procedure  which  has  proved 
satisfactory  in  the  hands  of  several  investigators.  Probably 
simpler  methods  will  be  devised  in  time,  but  this  appears  to  be  the 
best  thus  far  developed.  The  material  is  smeared  over  the  surfjice 
of  successive  tubes  of  scrum  agar  or  over  the  surface  of  this  medium 
in  Petri  dishes.  These  are  allowed  to  stand  for  several  days,  and 
the  colonies  which  develop  are  marked,  as  they  are  not  B.  abortus. 
The  plates  are  then  placed  in  a  desiccator,  whose  cubic  content 
of  air  has  been  determined,  and  plates  of  agar  thickly  seeded  with 
Bacillus  subtilis  are  introduced,  so  that  about  16  sq.  cm.  of  surface 


ABORTION    BACILLUS   GROUP 


343 


is  present  per  every  240  c.c.  of  space.  It  has  been  found  experi- 
mentally that  this  will  diminish  the  oxygen  pressure  to  a  point 
where  the  Bacillus  abortus  will  develop.  After  three  days  the 
plates  are  examined  and  search  made  for  the  B.  abortus  in  the 
spaces  between  other  colonies  on  the  original  plates.  This  same 
device,  of  reducing  oxygen  pressure  by  means  of  cultures  of  Bacil- 
lus subtilis,  may  be  used  in  the  study  of  growth-characters  on 
other  media.  The  organism  will  grow  in  agar  without  addition 
of  serum,  particularly  at  the  surface,  but  the  addition  of  serum  is 
decidedly  beneficial.  The  colonies  develop  at  the  surface  in  about 
three  days  at  37°  as  small,  usually  discrete,  transparent  dots.  In 
shake  cultures  in  serum  agar  they  appear  in  about  four  days  in 
a  well-defined  stratum  about  10  to  20  mm.  below  the  surface. 
The  individual  colonies  may  reach  a 
diameter  of  1  mm.  when  well  separated 
from  each  other.  Growth  in  gelatin 
at  room-temperature  is  slow.  Bouil- 
lon cultures  show  development  some 
millimeters  below  the  surface,  the 
medium  above  this  remaining  clear. 
Milk  is  not  coagulated.  Little  or  no 
growth  takes  place  on  potato^^- — 

Physiology. — The  optimum  growth 
temperature  is  37°,  although  the  organ- 
ism is  found  to  multiply  slowly  at 
room-temperature.  The  relationship 
to  oxygen,  which  has  already  been 

discussed,  classifies  the  B.  abortus  as  a  micro-aerophile  rather 
than  a  strict  anaerobe.  Strangely  enough,  Bang  reports  that  the 
organism  will  also  grow  in  an  atmosphere  of  pure  oxygen;  two 
oxygen  optima  are,  therefore,  evident.  It  is  relatively  resistant 
to  desiccation.  It  will  remain  alive  for  months  in  a  retained 
mummied  fetus,  and  for  a  year  or  more  in  a  culture-medium. 
Acids  and  gas  are  not  produced  from  carbohydrates. 

Pathogenesis. — Experimental  Evidence. — Bang  demonstrated 
the  etiologic  relationship  of  the  organism  to  the  disease  by  intra- 
venous injection,  and  by  injection  into  the  vagina  and  uterus  of 
pregnant  cows.  Preisz  did  not  succeed  in  transmitting  the  disease 


Fig.  142. — Bacillus  abor- 
tus. Culture  in  serum  agar 
showing  the  definite  stratum 
in  which  the  colonies  develop 

(Xowak). 


344  VETERINARY   BACTERIOLOGY 

by  vaginal  injections  into  the  cow,  guinea-pig,  or  rabbit.  Nowak 
succeeded  in  producing  typical  abortion  of  dead  feti  in  guinea- 
pigs  and  rabbits  by  intraperitoneal  and  intravenous  injections. 
He  did  not  succeed  by  intra vaginal  injections. 

Character  of  Disease  and  Lesions. — There  are  few  symptoms 
either  preceding  or  following  the  expulsion  of  the  fetus.  The 
infection  seems  to  result  in  the  death  of  the  fetus,  and  the  organism 
can  be  readily  isolated  in  pure  culture  from  such.  Bang  regards  the 
disease  as  a  specific  uterine  catarrh. 

Immunity. — It  is  known  that  cows  that  have  aborted  one  or 
more  times  may  become  immunized  against  the  disease.  Vaccina- 

.4     --,*  -; 

• '  'H   • 

^P  Mi       -jf 

' 


Fig.  143. — Bacillus  abortus,  colonies  on  serum  agar  (Nowak). 

tion  and  serum  treatments  have  been  attempted,  but  their  worth 
has  not  been  thoroughly  proved. 

Bacteriologic  Diagnosis. — Bacteriologic  diagnosis  may  be  made 
certain  by  the  isolation  of  the  specific  organism  in  culture  as 
outlined  above.  A  tentative  diagnosis  may  be  made  by  pre- 
paring stained  mounts,  and  demonstrating  the  presence  of  a  short, 
gram-negative  bacillus  in  the  uterine  exudate  and  in  the  blood 
and  tissues  of  the  fetus. 

Transmission. — The  disease  is  probably  most  frequently  trans- 
mitted by  the  bull.  Infection  may  also  occur  from  other  cows, 
from  infected  stables,  and  bedding. 


CHAPTER  XXXIII 

BACILLUS  NECROPHORUS  GROUP 

THIS  group  is  at  present  represented  by  a  single  species,  ac- 
cording to  most  investigators.  There  is  a  real  question  as  to  the 
proper  placing  of  this  form,  whether  among  the  true  bacteria  or 
in  the  group  of  Actinomyces.  As  will  be  observed  from  the  dis- 
cussion of  the  morphology,  the  organism  resembles  the  latter 
rather  more  than  true  bacilli.  There  has,  however,  been  no  satis- 
factory demonstration  of  branching,  although  some  of  the  forms 
seen  in  culture-media  suggest  that  such  may  occur. 

Bacillus  necrophorus 

Synonyms. — Bacillus  diphtheria  vitulorum;  B.  filiformis;  Strep- 
tothrix  cuniculi;  Actinomyces  cuniculi;  B.  necroseos;  Streptothrix 
necrophora. 

Disease  Produced. — A  large  number  of  diphtheritic  and  ne- 
crotic  pathologic  conditions  in  animals. 

Loffler,  in  1894,  described  this  organism  from  calf  diphtheria. 
Later,  Schiitz  found  it  associated  with  the  intestinal  ulcerations 
of  hog-cholera.  It  is  now  known  to  produce  disease  in  birds  and 
in  both  domestic  and  wild  animals. 

Distribution. — Bang  succeeded  in  demonstrating  the  presence 
of  this  organism  in  the  feces  of  normal  hogs,  but  not  in  the  in- 
testinal contents  of  the  cow.  It  is  probably  rather  widely  dis- 
tributed in  some  localities.  The  infection  has  been  described 
from  various  sections  of  Europe  and  America. 

Morphology  and  Staining. — The  organism  is  a  long,  slender  rod, 
usually  bent  more  or  less,  although  coccus-like  forms  and  fila- 
ments may  be  observed.  It  is  about  0.7  to  1.5  ^  in  diameter. 
In  the  tissues  and  colonies  the  filaments  are  matted  together, 
but  definite  branching  has  not  been  satisfactorily  demonstrated. 
The  stained  rods  are  usually  beaded.  Involution  forms,  as  long 

345 


346  VETERINARY   BACTERIOLOGY 

clubs,  frequently  occur.  The  organism  is  non-motile,  and  does  not 
produce  spores  or  capsules.  It  stains  readily  with  the  common 
anilin  dyes,  but  is  gram-negative. 

The  peculiarities  of  staining,  the  possession  of  easily  stained 
granules,  or  of  a  vacuolate  protoplasm,  have  caused  some  authors 
to  group  this  germ  with  the  diphtheria  bacillus,  but  these  ap- 
pearances are  even  more  characteristic  of  certain  of  the  Actino- 
myces,  particularly  those  isolated  from  soil. 

Isolation  and  Culture. — Bacillus  necrophorus  is  most  easily 
isolated  in  pure  culture  by  inoculating  infected  tissue  into  rabbits 


Fig.  144. — Bacillus  necrophorus  (Mohler,  Bureau  of  Animal  Industry,  Circular 

No.  160). 

or  white  mice.  The  pure  culture  may  then  be  secured  from  the 
infected  organs.  The  cultural  characters  are  all  modified  by  the 
fact  that  the  organism  is  a  strict  anaerobe. 

Colonies  may  develop  upon  the  surface  of  agar  plates  if  the 
oxygen  is  removed  by  the  alkaline  pyrogallate  method,  but  not, 
according  to  Mohler  and  Morse,  in  an  atmosphere  of  hydrogen 
or  in  a  vacuum.  They  appear  in  forty-eight  hours  as  minute, 
dirty  white,  round,  opaque  colonies,  with  gas-bubbles  developing 
below  the  surface.  In  seventy-two  hours  the  colony  appears 
wooly,  and  the  central  portion,  upon  microscopic  examination,  is 
shown  to  be  a  felted  mass  of  threads  with  a  border  of  long,  wavy 


BACILLUS  NECROPHORUS  GROUP  347 

filaments.     Bouillon  becomes  turbid,  and  then  gradually  clears, 
with  subsidence  of  the  organism.     Gelatin  is  not  liquefied. 

Physiology. — B.  necrophorus  is  an  obligate  anaerobe.  Its 
temperature  growth  limits  are  between  30°  and  40°,  with  an 
optimum  at  about  35°.  The  organism  is  readily  destroyed  by 
disinfectants.  No  pigment  is  produced.  A  very  characteristic 
odor,  "  between  the  odor  of  cheese  and  of  glue,"  may  be  noted  in 
both  cultures  and  lesions.  No  enzymes  capable  of  liquefying 
gelatin  or  blood-serum  are  produced.  Gas  is  formed  in  bouillon. 
Milk  is  not  coagulated,  nor  are  acids  formed.  Indol  is  produced. 

Pathogenesis. — Experimental  Evidence. — Rabbits  may  be  read- 
ily infected  with  the  B.  necrophorus.  A  subcutaneous  injection  of 
a  small  amount  of  necrosed  tissue  results  in  the  death  of  the  rabbit 
in  about  a  week.  The  inoculated  area  is  necrotic  to  some  depth, 
and  to  a  distance  along  the  surface  of  half  to  one  inch  from  the 
point  of  injection.  The  necrosis  is  complete,  and  the  tissues  wholly 
disintegrated.  In  some  cases  gas-bubbles  may  be  observed.  The 
inoculation  of  pure  cultures  results  in  death  more  slowly;  fre- 
quently two  weeks  are  required.  The  animal  dies  suddenly 
after  a  series  of  convulsions.  Mice  are  readily  infected.  Guinea- 
pigs  are  much  more  refractory,  but  occasionally  die  as  a  result 
of  inoculation.  There  seems  to  be  abundant  experimental  evi- 
dence to  connect  the  B.  necrophorus  with  many  types  of  necrosis 
in  animals.  There  is  no  evidence  that  the  organism  enters  the 
normal  healthy  unbroken  skin.  It  is  usually  a  secondary  invader. 

Character  of  Disease  and  Lesions  Produced. — Mohler  and 
Washburn  have  given  an  excellent  resume  of  the  conditions  under 
which  this  organism  has  been  found.  In  many  of  these  conditions 
it  has  not  been  satisfactorily  established  that  this  organism  is  the 
sole  cause,  for  pyogenic  cocci  and  other  organisms  may  produce 
the  same  changes.  More  work  is  needed  upon  these  infections. 
The  possible  presence  of  this  organism  in  necrotic  infections  of  all 
kinds  must  be  borne  in  mind.  The  organism  has  been  reported 
from  the  following  infections,  and  probably  in  most  cases  is  res- 
ponsible for  the  accompanying  necrosis:  necrotic  dermatitis, 
necrotic  scratches  in  the  horse,  necrotic  pox  in  horses,  cattle, 
goats,  and  hogs,  several  types  of  necrosis  in  rabbits,  necrosis  of  the 
hoof  in  the  horse,  necrosis  of  the  mouth  and  esophagus,  ulcerative 


348  VETERINARY   BACTERIOLOGY 

and  necrotic  vulvitis,  vaginitis,  and  metritis,  foot-rot  of  cattle, 
lip  and  leg  ulceration  of  sheep,  necrotic  omphalophlebitis  and 
joint  ill  in  young  animals,  necrosis  in  the  alimentary  tract  and 
other  viscera  in  many  animals,  and  possibly  even  avian  diphtheria. 
It  is  sometimes  of  considerable  economic  significance,  particularly 
in  the  so-called  lip  and  leg  ulceration  of  sheep.  Some  of  the  affec- 
tions, particularly  this  latter,  are  known  to  be  contagious.  Much 
work  still  remains  to  be  done,  however,  on  the  different  infections 
and  possible  variations  in  virulence. 

The  lesions  produced  in  all  tissues  have  many  common  charac- 
ters. They  are  essentially  coagulation  necroses  with  caseation. 
Metastatic  infection  is  very  apt  to  occur.  The  local  lesion  is 
described  by  Mohler  and  Morse  as  a  "  sharply  circumscribed 
patch  of  yellowish  or  dull  brown,  sometimes  greenish  white, 
homogeneous,  structureless,  dry,  crumbly  tissue  debris  of  soft, 
cheesy  consistence,  resembling  compressed  yeast,  and  manifesting  a 
characteristic  stench.  The  line  of  demarcation  between  the  living 
tissue  and  the  dead  mass  is  a  narrow  hyperemic  zone."  A  false 
membrane  is  formed  over  the  surface  as  a  "  result  of  coagulation 
necrosis  of  the  inflammatory  exudate  and  entanglement  in  its 
meshes  of  the  hyaline  degenerated  tissue-cells  and  leukocytes." 

Immunity. — It  has  been  supposed  that  the  organism  produces  a 
true  toxin  because  of  its  intense  local  destruction  of  tissue,  and 
because  of  the  death  of  laboratory  animals  with  many  of  the  symp- 
toms of  a  toxemia.  The  toxin  has  not  been  isolated,  however.  It 
is  stated  that  intravenous  injections  of  the  organism  into  the  goat 
confer  an  immunity.  No  practicable  method  of  immunization 
has  been  developed. 

Bacteriological  Diagnosis. — The  organism  may  be  observed  in 
mounts  prepared  from  the  tissue  just  surrounding  the  necrosed  area. 
Its  appearance  is  characteristic  enough  to  differentiate  it  from  other 
forms  that  may  be  present.  Animal  inoculations,  preferably  into 
the  rabbit,  are  generally  necessary  to  secure  pure  cultures. 

Transmission. — It  is  improbable  that  the  organism  ever  gains 
entrance  through  the  unbroken  skin  or  mucous  membrane. 
Scratches,  wounds,  abrasions,  or  injuries  of  other  types  supply  an 
infection  atrium.  The  disease  must  be  regarded  as  mildly  con- 
tagious, however. 


CHAPTER  XXXIV 

GROUP  OF  SPORE-BEARING  ANAEROBES 

THE  six  organisms  belonging  to  this  group  are  Bacillus  tetani, 
causing  tetanus;  B.  chauvcei,  of  blackleg;  B.  gastromycosis  ovis, 
of  bradsot;  B.  cedematis,  of  malignant  edema;  B.  welchii,  of  em- 
physematous  edema;  and  the  B.  botulinus,  of  meat-poisoning. 

The  organisms  of  this  group  are  united  because  of  their  lack 
of  tolerance  of  free  oxygen.  They  will  develop  in  the  presence  of 
small  amounts  of  oxygen,  but  not  with  an  oxygen  pressure  as 
great  as  that  of  the  atmosphere.  Morphologically,  these  organ- 
isms are  not  unlike.  All  are  bacilli  producing  spores.  Other 
characters,  such  as  retention  of  Gram's  stain  and  motility,  are 
inconstant.  The  members  of  this  group  may  be  differentiated 
from  each  other  in  most  cases  by  their  morphological  and  cultural 
characters,  although,  for  the  separation  of  some,  animal  experi- 
mentation is  necessary. 

Bacillus  tetani 

Synonym. — Bacillus  of  Nicolaier. 

Disease  Produced. — Tetanus  or  lockjaw  in  man  and  animals. 

Nicolaier,  in  1889,  observed  the  Bacillus  tetani  in  pus  from 
laboratory  animals  that  had  died,  following  subcutaneous  inocula- 
tion with  small  amounts  of  garden-soil.  He  cultivated  the  organ- 
ism, but  did  not  succeed  in  securing  it  in  pure  cultures.  Kitasato, 
in  1889,  succeeded  in  growing  the  organism  in  pure  culture,  and  in 
transmitting  the  disease  experimentally.  Kitasato  and  Veyl, 
in  1890,  described  the  production  of  the  tetanus  toxin. 

Distribution. — The  organism  is  found  in  all  parts  of  the  world. 
It  is  particularly  common  in  street-dust  and  fertilized  garden-soil, 
and  is  found  quite  constantly  in  the  alimentary  tract  of  herbivor- 
ous animals.  It  seems  probable  that  it  may  for  a  time  main- 
tain a  saprophytic  existence  and  multiply  in  the  soil. 

349 


350 


VETERINARY   BACTERIOLOGY 


Morphology  and  Staining. — B.  tetani  is  a  rather  long,  slender 
rod,  0.5  by  2  to  5  p,  with  rounded  ends,  usually  single,  rarely 
in  short  chains.  It  is  motile  by  means  of  numerous  peritrichic 
flagella.  Capsules  are  not  produced.  Spores  are  formed  abun- 
dantly. Their  size  and  position  are  so  characteristic  as  to  be  practi- 
cally diagnostic.  They  are  spherical,  two  or  three  times  the  diam- 
eter of  the  rod,  and  terminal,  giving  the  organism  the  appearance4 
of  a  drumstick.  The  organism  stains  readily  with  the  ordinary 
anilin  dyes  and  is  gram-positive. 

Isolation  and  Culture. — The  isolation  in  pure  culture  of  B. 
tetani  is  attended  with  considerable  difficulty,  largely  on  account 
of  its  being  an  obligate  anaerobe.  Kitasato  first  succeeded  in 

isolating  it  by  producing  tetanus 
in  experimental  animals,  then 
inoculating  broth,  and,  after 
growth  had  taken  place,  heating 
to  a  temperature  of  80°  for  half 
an  hour.  This  temperature 
should  destroy  all  but  spores. 
The  broth  may  then  be  inocu- 
lated into  agar  or  gelatin  and 
kept  under  anaerobic  condi- 
tions. If  spores  of  other  an- 
aerobes are  present,  it  may  be 
necessary  to  make  several  con- 
secutive animal  inoculations 
and  isolations. 

The  colonies  of  the  tetanus  bacillus  upon  gelatin  plates  show 
minute  radiating  lines  of  growth  from  a  central  nucleus,  resembling 
somewhat  those  of  B.  subtilis.  Gelatin  stabs  show  an  arborescent 
growth.  The  gelatin  is  slowly  liquefied.  Radiating  filaments 
are  also  produced  in  glucose  agar  stabs.  Bouillon  is  clouded,  and  a 
sediment  forms.  Blood-serum  is  liquefied.  Milk  is  coagulated, 
with  production  of  acid. 

Physiology. — The  optimum  growth  temperature  is  37.5°,  but 
the  organism  multiplies  rapidly  at  room-temperatures.  It  is  an 
obligate  anaerobe,  and  in  pure  culture  requires  practically  com- 
plete exclusion  of  oxygen.  It  will  develop,  however,  under  aerobic 


Fig.  145. — Bacillus  tetani,  rods  and 
spores  (Giinther). 


GROUP   OF   SPORE-BEARING    ANAEROBES  351 

conditions  when  in  mixed  cultures  with  aerobes.  The  spores  resist 
desiccation  indefinitely.  They  are  also  much  more  than  usually 
resistant  to  the  action  of  disinfectants.  Likewise,  resistance  to 
heat  is  so  marked  that  Theobald  Smith  found  in  one  case  that  ex- 
posure to  live  steam  for  seventy  minutes  failed  to  destroy  the  or- 
ganism, although  usually  a  shorter  period  suffices. 


. 

Fig.  146. — Bacillus  tetani,  colonies  Fig.    147 .— Bacillus    tetaniy    deep 

in    dextrose    gelatin    (Frankel    and  stab    culture    in    dextrose     gelatin 

Pfeiffer).  (Frankel  and  Pfeiffer). 

Acids  are  produced  from  carbohydrates,  and  a  small  amount 
of  gas  from  dextrose.  Enzymes  which  liquefy  gelatin  and  blood- 
serum  have  been  demonstrated. 

Pathogenesis. — Experimental  Evidence. — Injection  of  pure  cul- 
tures of  B.  tetani  into  experimental  animals  causes  the  develop- 
ment of  a  typical  tetanus.  The  white  mouse  is  among  the  most 
susceptible  of  animals  to  inoculation.  Infection  of  this  animal 
is  within  one  to  three  days  followed  by  tetanic  convulsions  and 
death.  Birds  are  not  ordinarily  susceptible  to  infection.  The 


352  VETERINARY  BACTERIOLOGY 

disease  is  most  common  in  man  and  in  the  horse,  although  no 
mammalia  are  immune.  The  disease  is  not  transmitted  by  in- 
gestion.  The  injection  of  the  characteristic  toxin  is  followed  by 
the  symptoms  of  the  disease. 

Character  of  Disease  and  Lesions  Produced. — The  disease  is  a 
typical  toxemia.  There  is  rarely,  if  ever,  a  general  invasion  of  the 
tissues.  The  organism  remains  localized  at  the  seat  of  inoculation, 
and  produces  the  toxin  which  brings  about  the  characteristic 
symptoms.  The  entrance  of  the  organism  into  a  wound  is  not 
always  followed  by  the  development  of  tetanus,  for  anaerobic 
conditions  must  obtain,  and  it  has  been  found  that  tetanus  spores 
entirely  freed  from  toxin  cannot  germinate  when  introduced  into 
the  tissues  in  moderate  numbers.  It  is  evident  that  the  organism 
has  little  initial  pathogenic  power.  Usually  considerable  amounts 
of  dirt  are  introduced  into  the  wound  simultaneously  with  the 
organism,  and  produce  proper  conditions  for  rapid  development. 
The  tetanus  toxin  produced  is  absorbed,  for  the  most  part,  by 
the  end-organs  of  the  motor  nerves,  and  travels  to  the  nerve-cells 
of  the  central  nervous  system  by  way  of  the  axis-cylinders  of  the 
peripheral  nerves.  Possibly  a  part  of  the  toxin  may  be  carried 
to  the  central  ganglion-cells  by  the  blood-stream.  The  inoculation 
period  noted  is  believed  to  be  due  to  the  time  required  for  the  toxin 
to  pass  along  the  nerves.  That  the  toxin  has  a  special  affinity  for 
nervous  tissue,  and  may  be  bound  by  it,  has  already  been  noted 
in  the  discussion  of  toxins  and  antitoxins.  The  period  of  incuba- 
tion in  man  averages  about  nine  or  ten  days.  In  the  horse  it 
varies  from  four  to  twenty  days.  Under  exceptional  conditions 
this  period  may  be  much  longer.  Mortality  is  over  90  per  cent, 
when  there  is  a  short  period  of  incubation,  and  over  50  per  cent, 
where  the  period  is  prolonged.  The  characteristic  symptom  in  all 
animals  is  a  tetanus  or  stiffening  of  the  muscles.  The  muscles  at 
the  site  of  inoculation  are  generally  the  first,  and,  in  mild  cases, 
they  may  be  the  only  ones,  affected.  In  the  horse  the  appearance 
of  the  tetanus  or  lockjaw,  the  retraction  of  the  eyes  and  protrusion 
of  the  nictitating  membrane,  spasmodic  contraction  of  other  mus- 
cles of  the  head,  and  those  of  other  parts  of  the  body,  arc  diagnostic. 
A  postmortem  examination  usually  shows  absence  of  gross  lesions. 
Certain  degenerative  changes  in  the  motor  cells  of  the  cord  may 


GROUP   OF   SPORE-BEARING   ANAEROBES  353 

be  observed  in  stained  sections.  Hemorrhages  in  different  organs 
are  an  inconstant  accompaniment  of  the  disease. 

Immunity. — The  toxin  of  the  tetanus  bacillus  is  produced  in 
artificial  media  as  well  as  within  the  body.  For  the  preparation  of 
antitoxin  the  organism  is  grown  in  bouillon,  under  anaerobic  con- 
ditions, in  an  atmosphere  of  hydrogen,  with  surfaces  of  the  medium 
covered  with  paraffin  or  paraffin  oil  or  with  oxygen  excluded  in 
some  other  manner.  After  incubation  for  a  period  of  one  or  two 
weeks  the  broth  is  filtered  through  porcelain.  The  toxin  may  be 
prepared  in  dried  form  by  precipitation  with  an  excess  of  am- 
monium sulphate.  After  standing  overnight  the  brown  scum  is 
removed  and  dried,  first  between  hardened  filter-papers,  then  in  a 
desiccator,  pulverized,  and  preserved  in  a  darkened  refrigerator. 
Various  methods  of  purification  have  been  devised,  such  that  a 
dried  toxin  may  be  prepared  of  which  0.00000025  gm.  will  prove 
quickly  fatal  to  a  white  mouse.  As  has  been  said,  this  toxin  has 
a  peculiar  affinity  for  the  cells  of  the  central  nervous  system.  Two 
poisonous  constituents  of  the  toxin  have  been  differentiated— 
tetanolysin,  which  lakes  the  red  blood-cells,  and  tetanospasmin, 
which  gives  rise  to  the  characteristic  tetanus  symptoms. 

In  the  preparation  of  antitoxin  the  unprecipitated  broth  is 
used.  The  smallest  amount  of  toxin  that  will  certainly  kill  a 
350  gm.  guinea-pig  in  three  to  four  days  is  taken  as  the  unit 
of  toxicity.  Increasing  amounts  of  the  toxin  are  injected  at 
intervals  into  a  horse.  The  blood-serum  of  the  immunized  horse 
contains  the  specific  antitoxin.  Many  methods  of  standardization 
of  the  antitoxin  have  been  used.  In  the  United  States  it  is  titrated 
by  guinea-pig  injections  against  a  standard  toxin,  sent  out  by  the 
Hygienic  Laboratory  of  the  Public  Health  and  Marine  Hospital 
Service.  It  is  used  in  both  human  and  veterinary  medicine, 
principally  as  a  prophylactic.  The  tetanus  antitoxin  has  not 
taken  the  place  in  the  treatment  of  tetanus  that  is  occupied  by  the 
antitoxin  specific  for  diphtheria  in  the  treatment  of  that  disease. 
It  seems  that  the  symptoms  of  the  disease  are  noted  only  after 
the  union  of  the  toxin  with  nerve-cells;  that  is,  after  much  of  the 
damage  has  already  been  accomplished.  The  injection  of  anti- 
toxin then  will  doubtless  neutralize  any  toxin  present  in  the  blood, 
but  cannot  remove  the  toxin  already  bound  to  the  nerve-cells. 
23 


354  VETERINARY    BACTERIOLOGY 

The  antitoxin  is  generally  injected  subcutaneously,  but  in  severe 
cases  intravenous,  intraneural,  and  intraspinal  injections  are 
made  to  insure  the  contact  of  antitoxin  with  the  toxin  present. 
Its  use  is  doubtless  indicated  in  all  cases.  As  a  prophylactic,  it 
has  been  found  quite  certainly  to  prevent  the  development  of 
tetanus  when  injected  before  the  appearance  of  symptoms.  In 
human  medicine  it  is  customary  to  make  injections  following  severe 
wounds,  into  which  dust  and  dirt  have  gained  entrance,  such  as 
Fourth-of-July  wounds.  The  same  may  be  said  with  reference 
to  severe  wounds,  nail-punctures,  and  similar  traumata  in  the 
horse. 

Bacteriological  Diagnosis. — The  organism  may  frequently  be 
recognized  in  stained  mounts  of  the  pus  from  the  wound.  The 
drumstick  shape  of  the  sporulating  organism  is  quite  characteristic. 
Isolation  in  pure  culture  and  animal  inoculation  may  also  be  used. 
The  symptoms  of  tetanus  are  so  distinctive,  however,  that  these 
methods  are  rarely  called  into  use. 

Transmission. — Tetanus  is  one  of  the  best  examples  of  a  non- 
contagious,  infectious  disease.  Infection  occurs  practically  in- 
variably directly  through  the  skin.  The  almost  universal  pres- 
ence of  the  organism  about  stables  renders  infection  easy.  Nail- 
punctures  are  particularly  apt  to  result  in  tetanus,  as  they  introduce 
the  organism  deep  into  the  tissues;  superficial  healing  and  exclu- 
sion of  air  quickly  take  place,  and  conditions  are  then  right  for 
rapid  multiplication.  It  should  again  be  emphasized  that  it  seems 
very  difficult  for  the  tetanus  bacillus  to  gain  a  foothold  and  pro- 
liferate, except  in  tissues  that  have  been  injured.  The  constant 
presence  of  these  organisms  in  the  intestines  does  not  produce 
disease.  So-called  cryptic  infections  are  not  of  uncommon  oc- 
currence, particularly  in  the  horse.  In  these  the  point  at  which 
the  organism  gains  entrance  to  the  body  is  not  known.  Usually 
this  comes  either  from  the  wound  having  healed  superficially,  so 
as  to  be  indistinguishable,  or  from  the  wound  having  born  origin- 
ally so  insignificant  as  to  have  escaped  notice.  Some  investiga- 
tors believe  that  the  organism  may  occasionally  «;am  nil  ranee  to 
the  blood-stream  from  the  intestines,  but  is  unable  to  produce  an 
infection,  except  when  it  lodges  in  tissue  traumatically  or  otherwise 
injured,  such  as  a  broken  bone  or  a  bruise. 


GROUP    OF   SPORE-BEARING    ANAEROBES 


355 


Bacillus  chauvaei 

Synonyms. — Bacillus  feseri;  B.  chauvei;  B.  chauveaui;  B. 
anthracis  xymptomatici. 

Diseases  Produced. — Blackleg,  symptomatic  anthrax,  quarter 
evil,  quarter  ill,  Rauschbrand,  charbon  symptomatique  in  cattle 
and  rarely  in  sheep  and  goats. 

Arloing,  Cornevin,  and  Thomas,  in  1880,  described  the  B. 
chauvcei  as  the  cause  of  blackleg,  and  proved  its  etiological  relation 
to  the  disease.  Kitasato  first  grew  the  organism  in  pure  culture. 
Grassberger  and  Schattenfroh  «_ 

have  shown  that  this  organ- 
ism, as  well  as  the  organism 
of  malignant  edema  still  to 
be  considered,  is  a  member 
of  the  ubiquitous  group  of 
anaerobic  butyric  acid  bacilli. 


Fig.   148.— Bacillus  chauvcei  (Kolle 
and  Wassermann) . 


Fig.  149. — Bacillus  chawwi,  colonies 
in  a  dextrose  gelatin  shake  culture 
(Frankel  and  Pfeiffer). 


Morphology  and  Staining. — B.  chauvcei  is  a  large  bacillus  with 
rounded  ends,  usually  single,  but  occasionally  in  pairs,  0.5  to  0.6 
by  3  to  5  |W.  It  is  motile  by  means  of  peritrichic  flagella.  Invo- 
lution forms,  consisting  of  greatly  enlarged  rods,  are  frequently 
encountered,  particularly  in  old  cultures.  Capsules  have  not  been 
demonstrated.  Spores  are  produced,  sometimes  central,  but  more 
frequently  near  a  pole,  rarely  quite  terminal.  They  are  oval  in 
shape,  and  are  not  generally  more  than  twice  the  diameter  of  the 


356  VETERINARY   BACTERIOLOGY 

rod.  These  characters  enable  one  readily  to  differentiate  it  from 
the  B.  tetani.  When  the  spores  are  central,  the  spindle-shaped 
swollen  cell  is  called  a  clostridium.  The  organism  is  easily  stained 
by  the  common  aqueous  anilin  dyes,  but  is  gram-negative.  This 
reaction  to  Gram's  stain  is  a  little  uncertain,  some  cultures  retain- 
ing the  stain  to  some  degree. 

Isolation  and  Culture. — The  organism  may  be  readily  isolated 
in  pure  cultures  from  the  tissues  infected.  Plates  may  be  poured 
and  kept  under  anaerobic  conditions.  The  colonies  are  spherical, 
or  somewhat  irregular,  with  microscopic  radiations.  Dextrose 
gelatin  is  an  exceptionally  favorable  medium.  In  a  shake  culture 
the  colonies  appear  in  the  lower  portion  of  the  tube,  each  usually 
with  its  gas-bubble,  and  surrounded  by  a  liquefied  area.  Bouillon 
is  clouded,  gas  is  produced,  and  a  flaky  white  deposit  forms.  The 
reaction  in  milk  is  somewhat  variable;  according  to  most  authori- 
ties, it  produces  acid,  coagulates,  and  later  proteolyzes  the  casein. 

Physiology. — The  optimum  growth  temperature  is  about  blood- 
heat,  but  good  growth  occurs  at  room-temperatures.  The  organ- 
ism is  a  strict  anaerobe.  Concerning  its  other  physiological  char- 
acters, there  is  considerable  disagreement  among  investigators. 
This  may  be  due  to  the  fact  that  there  are  strains  which  react  very 
differently,  and  may  constitute  distinct  varieties.  Grassberger 
and  Schattenfroh  claim  that  the  organism  shows  considerable 
variability,  and  that  certain  characters  are  easily  lost.  The 
spores  are  quite  resistant  to  desiccation.  Heating  for  some  hours 
at  100°  is  necessary  certainly  to  destroy  them.  Gas  is  produced 
from  carbohydrates,  and  probably  also  from  proteins.  According 
to  some  authorities,  butyric  acid  is  produced. 

Pathogenesis. — Experimental  Evidence. — Inoculation  of  pure 
cultures  into  laboratory  animals  results  in  death,  with  production 
of  many  of  the  characteristic  symptoms  of  blackleg,  particularly 
the  edema  about  the  point  of  inoculation.  The  disease  may  also 
be  produced  in  cattle,  so  that  there  is  no  doubt  as  to  the  etiological 
relationship  of  this  organism  to  the  disease. 

Character  of  Disease  and  Lesions  Produced. — Blackleg  in  cattle 
is  characterized  by  a  swelling,  edema,  and  emphysema  of  the 
muscles  and  the  subcutaneous  tissues  of  the  infected  part.  Infec- 
tion appears  most  commonly  in  the  shoulder  or  hindquarter. 


GROUP   OF   SPORE-BEARING   ANAEROBES  357 

The  swelling  increases  rapidly  in  size,  and  the  emphysema  soon 
manifests  itself  by  the  crackling  sound  produced  when  the  thumb 
is  drawn  firmly  across  the  part.  After  death  the  organisms  con- 
tinue to  grow  and  the  body  becomes  distended  with  gas.  The 
subcutaneous  tissues  of  the  infected  part  are  edematous,  even 
gelatinous,  with  blood  and  gas-bubbles.  The  underlying  muscles 
are  dark  brown  or  even  blackish,  whence  the  name,  blackleg. 
The  disease  usually  results  fatally  in  cattle  in  from  one  to  three 
days  after  the  first  appearance  of  the  lesions. 

Immunity. — The  production  of  true  toxins  by  Bacillus  chauvcei 
is  not  well  understood.  According  to  some  authors,  no  toxin  can 
be  demonstrated.  Others  believe  that  there  is  a  relatively  thermo- 
stabile  toxin  produced  which  will  endure  a  temperature  of  even 
115°.  Grassberger  and  Schattenfroh,  who  have  made  the  most 
careful  study  of  this  problem,  have  succeeded  in  producing  broth 
cultures  containing  a  toxin  that,  in  doses  as  small  as  those  em- 
ployed with  diphtheria  toxin,  will  kill  laboratory  animals.  This 
toxin  is  produced  by  certain  strains  of  the  organism  only,  but  these 
they  believe  are  the  more  pathogenic.  This  toxin  they  have  shown 
to  be  thermolabile.  They  have  worked  out  methods  of  standardi- 
zation closely  resembling  those  of  Ehrlich  for  diphtheria.  Anti- 
toxin may  be  produced  by  the  injection  of  increasing  doses  into 
suitable  animals,  particularly  cattle,  and  this  antitoxin  has  a 
protective  influence  when  injected  into  other  animals.  This 
method  of  immunization  has  never  come  into  general  use. 

Animals  that  have  recovered  from  an  attack  of  the  disease 
acquire  immunity  to  a  recurrence.  Very  young  cattle  and  aged 
cattle  have  a  considerable  degree  of  natural  immunity.  To 
what  the  immunity  developed  may  be  due  is  not  well  understood. 
Probably  it  is  in  part  opsonic. 

Active  immunization  of  animals  is  extensively  practised. 
Various  methods  of  attenuation  of  the  organism  for  the  vaccine 
have  been  developed.  That  in  common  use  in  the  United  States 
is  the  one  adopted  by  the  Bureau  of  Animal  Industry,  and  is 
essentially  that  developed  by  Kitt.  Fresh  material  is  secured 
by  macerating  in  a  mortar  the  muscle  tissue  from  a  blackleg 
tumor  and  squeezing  the  fluid  through  a  linen  cloth.  This  is 
spread  in  a  thin  layer,  and  dried  to  a  brown  scale  at  a  temperature 


358  VETERINARY    BACTERIOLOGY 

/• 

at  about  blood-heat.  This  dried  virus  retains  its  virulence  for 
several  years,  at  least.  The  vaccine  is  prepared  by  mixing  one  part 
of  this  material  with  two  parts  of  water  and  placing  in  a  hot-air 
oven  at  a  temperature  of  95  °  to  99  °  for  six  hours.  This  dries  the 
material  and  attenuates  the  organism.  It  is  then  pulverized  and 
put  up  in  packages  containing  a  definite  number  of  doses.  Before 
use,  a  cubic  centimeter  of  water  for  each  dose  is  added,  and  the 
material  mixed  and  then  filtered.  The  injection  then  is  made 
with  1  c.c.  The  dried  material,  pressed  into  the  form  of  tablets, 
is  sometimes  inserted  under  the  skin  without  suspending  it  in 
water.  Vaccination  has  proved  quite  satisfactory. 

Bacteriological  Diagnosis. — A  presumptive  determination  of 
the  organisms  may  be  made  by  smear  preparations  from  the  in- 
fected tissues.  Anaerobic  cultures  will  demonstrate  the  specific 
organism  in  pure  culture  if  inoculated  with  a  bit  of  the  tissue 
before  decomposition  has  begun  and  putrefactive  bacilli  have4 
gained  entrance.  Animal  inoculation,  particularly  subcutaneous 
inoculation  into  the  guinea-pig,  may  prove  useful.  Usually  the 
symptoms  of  the  disease  are  so  characteristic  that  a  bacteriological 
test  is  wholly  unnecessary. 

Transmission. — It  is  believed  that  B.  chauvcei  is  widely  dis- 
tributed in  nature.  The  disease  occurs  only  in  certain  localities. 
There  are  districts  which  are  never  affected.  Attempts  have  been 
made  to  correlate  the  topography  of  the  country,  such  as  character 
of  soil,  presence  of  marsh  land,  etc.,  with  the  prevalence  of  the 
disease,  but  without  much  success.  Organisms  closely  related  to 
B.  chauvcei  may  be  found  widely  in  the  soil,  but,  for  the  most  part, 
they  do  not  possess  the  peculiar  pathogenic  characters  of  this  form. 
Infection  is  believed  to  occur  through  wounds.  The  disease  is 
rarely,  if  ever,  contracted  directly  by  one  animal  from  another.  It 
is  a  non-contagious  infectious  disease.  It  is  not  always  possible  to 
locate  the  point  at  which  the  organisms  gained  entrance — in  fact, 
these  cryptic  infections  constitute  a  considerable  proportion  of  the 
rases.  It  is  possible  that  the  explanation  sometimes  offered  for 
similar  infections  in  tetanus  will  hold  good  here  also;  that  is,  that 
the  organisms  may  occasionally  gain  entrance  to  the  blood  from  the 
intestinal  tract,  and  that  they  cannot  produce  disease  except  when 
they  lodge  in  some  tissue  that  has  been  injured,  as  from  a  bruise. 


GROUP   OF   SPORE-BEARING    ANAEROBES 


359 


Bacillus  gastromycosis  ovis 

Disease  Produced. — Bradsot  or  braxy  in  sheep. 

An  organism  morphologically  quite  identical  with  the  Bacillus 
chauvcei  has  been  isolated  and  claimed  to  be  the  cause  of  braxy  or 
bradsot  in  sheep.  The  disease  is  known  principally  from  northern 
Europe  and  the  British  Isles.  There  are  certain  chemical  and 
pathogenic  characters  of  this  organism  which  seem  to  separate  it 
from  the  Bacillus  chauvcei.  Careful  comparative  studies  of  these 
organisms  are  needed. 

Bacillus  oedematis 

Synonyms.—  Vihrion  septique;  Bacillus  cedematis  maligni. 

Diseases  Produced. — Malignant  edema;  Malignes  (Edem, 
(pdeme  malin,  septicemie  gangreneuse,  in  various  animals  and 
in  man. 

Pasteur,  in  1877,  found  that  the  injection  of  putrid  flesh  into 
a  rabbit  was  followed  by  an  edema  at  the  point  of  inoculation, 
and  ultimately  by  the  death 
of  the  animal,  with  changes 
in  many  of  the  internal 
organs.  That  these  changes 
were  due  to  a  specific  organ- 
ism, and  not  to  the  poisons 
of  the  putrid  flesh  alone, 
was  shown  by  transfers  from 
one  animal  to  another,  and 
by  the  isolation  of  an  anaero- 
bic bacterium.  Koch  later 
(in  1881)  studied  the  disease. 

Morphology  and  Stain- 
ing.— The  Bacillus  cedenmti* 
closely  resembles  the  B. 
chauvcei  morphologically,  and 

is  apparently  closely  related  to  it.  Some  investigators  believe 
that  the  two  organisms  are  simply  two  varieties  of  the  same 
species.  The  organism  is  a  rod,  0.8  to  1  by  2  to  10  ^/,  with 
rounded  ends,  single  or  in  chains.  Many  of  the  cells  are  long 
and  filamentous.  It  is  motile,  with  numerous  peritrichic  flagella. 
( 'apsules  have  not  been  demonstrated.  Spores  are  produced, 


Fig.  150. — Bacillus  cedematis,  spores 
and  rods  from  an  agar  culture  (Frankel 
and  Pfeiffer). 


360  VETERINARY   BACTERIOLOGY 

usually  equatorial,  but  sometimes  polar.  The  rod  is  not  greatly 
distended  by  the  spore,  although  the  snowshoe  or  clostridium 
shape  is  usually  evident.  The  organism  is  gram-negative,  and 
stains  readily  with  the  common  anilin  dyes. 

Isolation  and  Culture. — The  organism  may  be  secured  in  pure 
cultures  without  difficulty,  under  anaerobic  conditions,  from  the 
edematous  tissues  of  the  infected  animal.  It  may  usually  be 
isolated  from  garden-soil  by  inoculation  of  a  guinea-pig  or  a  rabbit. 
Its  cultural  characters  do  not  differ  from  those  described  for 
Bacillus  chauvcei.  Gelatin  and  blood-serum  are  digested,  milk  is 
curdled,  and  the  casein  digested. 


Fig.   151. — Bacillus  cedematis,    tissue   smear    showing   rods   without   spores 
(Frankel  and  Pfeiffer). 

Physiology. — Growth  is  luxuriant  at  room-temperatures,  as  well 
as  at  blood-heat.  The  spores  are  resistant  to  desiccation  and  to 
heat.  Gas  is  produced  from  dextrose,  probably  also  from  pro- 
teins. Enzymes  that  liquefy  gelatin,  blood-serum,  and  casein 
are  present. 

Pathogenesis. — Experimental  Evidence. — Inoculation  of  pure 
cultures  of  the  organism  into  the  laboratory  animals,  and  also  into 
the  horse  and  other  domestic  animals,  will  produce  a  typical  infec- 
tion. 

Character  of  Disease  and  Lesions  Produced. — The  tissues 
at  the  point  of  invasion  are  distended  with  gas-bubbles  and  are 
infiltrated  with  yellow  or  red  serum.  The  muscle  becomes  dark 


GROUP   OF   SPORE-BEARING   ANAEROBES 


361 


and  brittle.  Hemorrhages  are  generally  to  be  found  in  the  sub- 
cutaneous tissues.  The  disease  has  been  noted  in  man,  and  is 
not  uncommon  in  the  horse  and  the  sheep.  It  occurs  more  rarely 
in  cattle.  It  is  a  typical  wound  infection. 

Immunity. — Animals  which  recover  from  an  infection  are  found 
to  be  thereafter  immune.  The  organism  is  also  known  to  produce 
a  leukocidin,  which  destroys 
white  blood-cells.  Aside  from 
these  facts,  little  is  known  rela- 
tive to  the  factors  determining 
immunity  in  this  disease. 

Transmission. — The  organ- 
ism usually  gains  entrance 
through  wounds,  although  the 
possibility  of  a  cryptic  infec- 
tion, such  as  is  claimed  to  occur 
in  tetanus,  should  not  be 


Fig.  152. — Bacillus  cedematis,  dextrose 
gelatin  culture  (Giinther). 


ignored.  In  man  the  disease 
has  been  known  to  occur  fol- 
lowing injections  in  which  an 
unclean  hypodermic  syringe  was  used,  and  a  case  has  been  reported 
in  which  the  organisms  were  believed  to  have  gained  entrance  to  the 
body  through  the  intestinal  ulcers  of  typhoid  fever.  Infection 
may  follow  delivery,  castration,  shearing  of  sheep,  use  of  unclean 
syringes  or  instruments,  or  dirty  wounds  of  any  kind. 

Bacillus  welchii 

Synonyms. — Bacillus  aerogenes  capsulatus;  B.  phlegmones  em- 
physematosce ;  B.  enteritidis  sporogenes;  Bacterium  welchii;  B. 
perfringens;  Granulobacillus  saccharobutyricus  immobilis;  B.  anaero- 
bicus  cryptobutyricus ;  B.  cadaveris  butyricus;  B.  emphysematis 
vagina?. 

Disease  Produced. — Gaseous  edema  in  man. 

Welch,  in  1892,  described  Bacillus  aerogenes  capsulatus  from 
the  body  of  a  man  who  died  from  an  aortic  aneurysm.  The  in- 
ternal organs  and  subcutaneous  tissues  showed  considerable 
emphysema.  Since  that  time  it  has  been  repeatedly  isolated  in 
Europe  and  America. 


302  VETERINARY    BACTERIOLOGY 

Distribution. — The  organism  is  common  in  garden-soil,  par- 
ticularly that  contaminated  with  excreta. 

Morphology  and  Staining. — B.  welchii  is  a  rod,  1  by  3  to  6  /w, 
with  rounded  ends  when  single  or  truncate  when  in  chains.  It 
frequently  occurs  in  chains,  but  may  be  found  in  pairs  and  small 
groups.  It  is  non-motile.  In  this  respect  it  differs  from  the  other 
members  of  this  group.  Spores  are  produced  only  under  certain 
conditions.  They  are  developed  best  upon  the  surface  of  blood- 
serum  in  anaerobic  cultures.  They  are  central,  and  the  cells 
develop  as  clostridia.  Capsules  may  be  demonstrated  in  the  body 
fluids  and  in  some  artificial  media.  The  organism  stains  readily 

with  the  common  anilin  dyes 
and  is  gram-positive.  Involu- 
tion forms  are  frequent  in  arti- 
ficial media. 

Isolation     and      Culture. — 

^  ;  McCampbell    has    described   a 

modification  of  Welch's  method 
of  isolation  as  follows:  "  1  gm. 

|  *s  ,T-  of  soil  is  shaken  in  sterile  NaCl 

solution   (0.85   per  cent.),  and 

%  «••  inoculated  into  sterile   neutral 

litmus    milk   tubes,   which   are 

Fig.  153. — Bacillus  welchii  (Jordan),      covered  with  25  mm.  of  neutral 

paraffin  oil,  for  the  purpose  of 

securing  anaerobiosis,  and  then  incubated  for  twenty-four  hours 
at  37°.  At  the  end  of  this  time  the  milk  in  the  tubes  is  coagu- 
lated and  shows  acids  and  gas.  A  subculture  is  made  in  a  second 
litmus  milk  tube  under  oil,  and  incubated  for  twelve  hours  in 
order  to  prevent  the  possible  overgrowth  of  other  bacteria.  At 
the  end  of  this  time  the  milk  usually  shows  coagulation,  acid-  and 
gas-production,  as  in  the  first  instance  ("stormy  fermentation"); 
0.5  c.c.  of  the  whey  in  the  subculture  is  then  injected  into  the 
posterior  auricular  vein  of  a  rabbit.  In  three  or  four  minutes 
the  animal  is  killed  by  a  blow  on  the  head,  and  the  body  is  in- 
cubated at  37°  for  eight  to  ten  hours,  at  the  end  of  which  time 
the  abdomen  is  markedly  distended  with  gas.  Wrhen  ignited,  this 
explodes  and  burns  with  a  hydrogen  flame.  The  thorax  of  the 


GROUP    OF    SPORE-BEARING    ANAEROBES  363 

animal  is  carefully  opened,  and  cultures  made  from  the  heart 
blood  in  dextrose  broth,  covered  by  neutral  paraffin  oil.  In 
from  eight  to  twenty-four  hours  the  culture  tubes  show  a  marked 
cloudiness,  abundant  gas-production,  and,  in  most  instances,  an 
odor  of  butyric  acid." 

The  colonies  upon  agar  and  gelatin  plates  are  round,  grayish, 
semitranslucent;  the}'  are  usually  nucleated,  and  resemble  those 
of  B.  tetani.  Upon  agar  slants  a  thin,  coalescent,  yellowish-white 
growth  occurs.  Gelatin  may  or  ma}'  not  be  slowly  liquefied. 
Bouillon  is  clouded  with  a  "heavy  precipitate.  Little  or  no  growth 
occurs  on  potato.  Upon  blood-serum  the  growth  resembles  that 
upon  agar.  There  is  some  liquefaction  along  the  line  of  inoculation. 
Milk  is  quickly  coagulated,  with  gas-  and  acid-production. 
.  Physiology. — Growth  occurs  best  at  37°.  The  thermal  death- 
point  for  non-sporulating  culture  is  50°  for  ten  minutes,  for  spores 
100°  for  fifteen  minutes.  Gas  is  produced  from  dextrose,  lactose, 
and  saccharose,  but  not  from  mannite.  Probably  some  differences 
are  to  be  found  in  various  strains.  Gas  is  likewise  produced  from 
pure  proteins,  such  as  recrystallized  egg-white.  The  gas  formula 

TT  9—^ 

is   approximately   ^Q  =    -.   .     Butyric,   lactic,   and   acetic   acids 

have  been  detected. 

Pathogenesis. — Experimental  Evidence. — Intravenous  injection 
of  the  rabbit  frequently,  though  not  always,  causes  death,  but 
subcutaneous  inoculations  are  without  effect.  Guinea-pigs  are 
susceptible,  as  are  also  pigeons. 

Character  of  Disease  and  Lesions  Produced. — Infections  with 
B.  welchii  among  the  lower  animals  have  been  noted  in  few  in- 
stances only,  and  then  only  in  the  rabbit  and  in  the  dog,  as  a  result 
of  severe  injuries.  However,  it  may  quickly  invade  tissues  after 
death,  and  give  opportunity  for  mistaken  diagnosis.  It  has  not 
been  shown  satisfactorily  that  it  ever  invades  the  tissues  general!}' 
before  death.  It  is  a  secondary  invader  in  practically  every  in- 
stance of  natural  infection.  It  has  been  found  in  emphysema  of 
many  organs  in  the  human  body.  Herter  believes  that  the  pres- 
ence of  large  numbers  of  this  organism  or  its  varieties  in  the  in- 
testines is  responsible  for  the  production  of  primary  pernicious 
anemia,  particularly  in  children. 


364  VETERINARY   BACTERIOLOGY 

From  the  veterinary  standpoint,  the  organism  is  of  principal 
interest,  not  so  much  because  of  its  slight  pathogenic  power,  as 
the  fact  that  it  may  be  confused  upon  isolation  with  other  spore- 
bearing  anaerobes. 

Immunity. — McCampbell  has  found  that  the  nitrates  of 
B.  welchil  grown  in  dextrose  bouillon  are  toxic,  but  showed  the 
toxicity  to  be  due  to  the  butyric  acid  produced.  No  true  toxin 
nor  endotoxin  could  be  demonstrated.  The  acids  are  also  hemo- 
lytic  and  leukocytotoxic.  Opsonins  are  present  in  normal  and 
in  increased  quantities  in  immune  sera,  as  are  also  specific  bacteri- 
cidal substances.  No  method  of  systematic  immunization  has 
been  developed,  nor  does  the  slight  pathogenicity  of  the  organism 
make  this  advisable. 

Bacteriological  Diagnosis. — The  organism  can  be  recognizeji 
certainly  from  tissues  only  by  isolation  and  morphological  examina- 
tion. Its  gram-positive  staining  characters,  lack  of  motility,  and 
the  difficulty  with  which  spores  may  be  demonstrated  are  charac- 
teristic. 

Transmission. — The  organism  probably  gains  entrance  to  the 
body  through  wounds  or  invades  the  tissues  after  death  from  the 
intestines. 

Bacillus  botulinus 

Disease  Produced. — Meat-  or  sausage-poisoning  in  man, 
botulism  (botulus  =  sausage). 

Van  Ermengem,  in  1896,  isolated  an  organism  from  sausage, 
which  he  believed  to  be  the  cause  of  poisoning.  The  organism  has 
since  that  time  been  several  times  isolated,  and  is,  therefore,  of 
some  hygienic  importance,  particularly  in  meat  inspection  and  in 
meat  hygiene.  This  disease  or  poisoning  should  not  be  confused 
with  that  produced  by  the  Badllus  enteritidis,  which  has  already 
been  discussed. 

Distribution. — Only  a  few  well-authenticated  reports  of  the 
isolation  of  the  organism  are  on  record,  and  these  principally 
from  European  countries. 

Morphology  and  Staining. — Bacillus  botulinus  is  a  large  bacil- 
lus, with  usually  rounded  ends,  0.9  to  1.2  by  4  to  6  p.  It  is  com- 
monly single  or  in  pairs,  sometimes  in  short  chains.  Involution 
forms  frequently  occur.  It  is  motile  by  means  of  four  to  eight 


GROUP   OF   SPORE-BEARING   ANAEROBES  365 

peritrichic  flagella.  Capsules  have  not  been  demonstrated.  Oval 
spores,  somewhat  greater  in  diameter  than  the  bacillus,  are  pro- 
duced at  the  poles.  The  organism  stains  readily  with  the  anilin 
dyes  and  is  gram-positive. 

Isolation  and  Culture. — The  colonies  on  dextrose  gelatin  are 
at  first  circular,  transparent,  light  yellow,  and  soon  liquefy  the 
gelatin.  Under  the  low  power  of  the  microscope  they  appear  to 
consist  of  granules  in  constant  motion.  Later  the  colonies  become 
brown  and  opaque.  Milk  is  not  curdled. 

Physiology. — The  organism  is  an  obligate  anaerobe.  Its 
optimum-growth  temperature  is  18°  to  20°.  It  grows  little,  if 
at  all,  at  blood-heat,  and  when  developing  at  this  temperature, 


Fig.  154. — Bacillus  botidinus  (van  Ermengem  in  Kolle  and  Wassermann) . 

produces  numerous  involution  forms.  Gas  is  produced  from  dex- 
trose, but  not  from  saccharose  or  lactose.  Acid,  in  part  butyric,  is 
produced  in  dextrose  media. 

Pathogenesis. — Injections  of  the  organism  into  the  body  of 
laboratory  animals  have  revealed  the  fact  that  the  organism  is 
pathogenic  only  by  virtue  of  the  toxins  that  are  elaborated  out- 
side of  the  body.  It  does  not  increase  in  numbers  in  the  tissues. 
Probably  this  may  in  part  be  accounted  for  by  its  normal  optimum- 
growth  temperature.  The  toxin  produced,  on  the  other  hand,  is 
very  poisonous,  whether  injected  or  ingested.  The  use  of  raw  or 
imperfectly  cooked  animal  foods  may  give  rise  in  man  to  the 
symptoms  of  botulism  in  the  course  of  twenty-four  to  thirty-six 
hours,  often  with  fatal  termination. 


366  VETERINARY    BACTERIOLOGY 

Immunity. — The  toxin  produced  by  Bacillus  botulinus  is  among 
the  most  powerful  known — 0.00005  to  0.0001  gm.  is  fatal  in 
three  to  four  days  when  injected  subcutaneously  into  a  guinea-pig, 
and  0.0001  to  0.0005  gm.  will  destroy  a  rabbit.  A  most  striking 
characteristic  of  this  toxin,  and  one  which  distinguishes  it  from 
those  of  diphtheria  and  tetanus,  is  its  ability  to  produce  poisoning 
when  taken  into  the  body  by  way  of  the  alimentary  tract.  Guinea- 
pigs,  and  even  apes,  are  killed  by  the  ingestion  of  0.01  c.c.  of  a 
dextrose  broth-culture  solution  in  which  the  organism  has  been 
grown.  The  toxin  is  destroyed  by  exposure  to  light  and  air. 
Hoating  to  a  temperature  of  80°  renders  it  non-toxic.  Antitoxin 
has  been  prepared  from  the  goat  and  from  the  horse  by  gradually 
increasing  doses  of  the  toxin.  This  antitoxin  exerts  both  a  pro- 
phylactic and  a  curative  effect  when  injected.  The  poisoning 
by  B.  botulinus  is  so  infrequent,  however,  that  the  antitoxin  is 
only  of  scientific  value. 

Bacteriological  Diagnosis. — This  can  be  accomplished  only  by 
isolation  and  cultivation  of  the  specific  organism. 

Transmission. — The  organism  has  been  isolated,  not  only 
from  poisonous  meat,  but  from  normal  swine  feces  as  well.  The 
disease  can  be  produced  only  by  the  ingestion  of  proteins  in  which 
the  organism  has  been  growing. 


CHAPTER  XXXV 

VIBRIO  OR  CHOLERA  SPIRILLUM  GROUP 

ONE  organism,  pathogenic  for  animals,  Spirillum  metchnikovi  „ 
and  one  pathogenic  for  man,  Spirillum  cholerce,  and  numerous 
non-pathogenic  forms  isolated  from  various  sources  have  been 
described  as  belonging  to  this  group. 

The  organisms  of  this  group  are  more  or  less  bent  rods,  usually 
only  a  segment  of  a  spiral,  rarely  showing  one  or  more  complete 
turns.  All  are  motile,  aerobic,  gram-negative,  and  without  spores. 

Spirillum  metchnikovi 

Synonyms.  —  Vibrio  metchnikovi;  Microspira  metchnikovi. 

Disease  Produced.  —  Septicemia  of  fowls. 

Gamaleia,  in  1888,  described  this  organism  from  an  epizootic  of 
fowl  septicemia  which  occurred  in  Russia.    What  appears  to  be  the 
same  organism  was  later  (1894) 
isolated  by  Pfuhl  from  water.  -^^,  ,  ? 

Distribution.  —  This  organism  'C  *-<<*•   ^%?>       ( 

does   not    seem   to   have   been  ^cVici 

isolated    from   such    a    disease  f  £j  % 

by  any   investigator  since  the  ^     *  v£ 

original     work     of     Gamaleia.       £    ^  _     -  ^  --, 


This  latter,  however,  was  suf-  &* 

\       '*  ,   ^         *»-T5^^ 

ficient  to  establish  its  etiologic     *%          ^^J^'      \*  ";  s  ^  M  •* 
relation  to  the  disease.     It   is  \*        f^      A  ^r^^\ 

possible    that    the    disease    is 

/  /  "^ 

more  widely  spread    than    the  ;   ,-    d* 

literature  would  indicate,  inas-     Fig      l55^s^riUum     metchnikon 
much  as  poultry  diseases  are  in  (Gimther). 

need  of  careful  investigation. 

Morphology  and  Staining.  —  It  is  a  curved  rod,  with  rounded 
ends,  or  sometimes  a  spiral  filament.     It  is  actively  motile  by  a 


368  VETERINARY    BACTERIOLOGY 

single  terminal  flagellum,  and  is  about  0.5  by  2  p.    Neither  capsules 
nor  spores  are  produced.     It  stains  readily,  but  is  gram-negative. 

Isolation  and  Culture. — The  organism  may  be  isolated  from  the 
intestinal  contents  of  adult  fowls  and  from  the  blood  of  younger 
fowls  by  plate  cultures  in  nutrient  gelatin.  Upon  these  plates 
small  white,  punctiform  colonies  are  developed  in  the  course  of 
twelve  to  sixteen  hours;  these  enlarge  rapidly,  cause  liquefaction 
of  the  gelatin,  and  are  soon  to  be  found  in  saucer-shaped  depressions 
in  the  medium.  Growth  in  a  stab  culture  in  gelatin  is  likewise 
rapid,  and  the  gelatin  is  quickly  liquefied  in  the  form  of  a  funnel. 
Upon  agar  slants  a  yellowish  layer  is  developed.  Bouillon  is 


Fig.  156. — Spirillum  metchnikovi  in  the  blood  of  a  pigeon  ( X  1000)  (Frankel 

and  Pfeiffer). 

clouded,  and  a  delicate  pellicle  may  form.  Milk  is  coagulated 
with  acid  reaction  and  without  solution  of  the  casein. 

Physiology. — The  organism  is  aerobic.  It  develops  almost  as 
rapidly  at  room-temperature  as  at  blood-heat.  The  thermal 
death-point  is  50°  for  five  minutes.  It  is  sensitive  to  the  pres- 
ence of  acids  in  culture-media,  and  soon  dies  in  consequence  of 
their  production  in  milk.  It  produces  acid  from  dextrose  and 
lactose,  but  no  gas.  Indol  is  formed.  Gelatinase  is  developed, 
but  no  enzyme  which  will  digest  casein. 

Pathogenesis. — Experimental  Evidence. — Subcutaneous  inocu- 
lations into  the  chicken,  pigeon,  and  guinea-pi^  ;iro  quickly  fatal; 
rabbits  and  mice  succumb  only  to  large  doses.  The  disease  ap- 


VIBRIO    OR    CHOLERA    SPIRILLUM    GROUP  369 

pears  as  a  septicemia.  According  to  Gamale"ia,  it  may  be  com- 
municated to  chickens  by  feeding  pure  cultures. 

Character  of  Disease  and  Lesions. — Chickens  affected  show 
many  of  the  same  symptoms  as  those  infected  with  chicken  cholera, 
except  that  the  temperature  is  little,  if  at  all,  elevated.  Diarrhea 
is  constantly  present.  There  is  a  marked  hyperemia  of  the  ali- 
mentary canal,  and  a  blood-tinged,  grayish-yellow  liquid  is  found 
in  the  small  intestine. 

Immunity. — No  true  toxin  has  been  demonstrated.     Agglutin- 


Fig.  157. — Spirillum  metchnikovi,  gelatin  stab  (Frankel  and  Pfeiffer 

ins  are  present  in  immune  serum.  Immunity  may  be  established 
in  animals  by  repeated  injections  of  killed  cultures. 

Bacteriological  Diagnosis. — This  may  be  made  on  the  basis 
of  microscopic  findings  and  isolation  in  pure  culture. 

Transmission. — The  disease  is  probably  transmitted  by  the 
ingest  ion  of  food  soiled  by  droppings  from  infected  fowls. 

Spirillum  choleras 

Synonyms. — Spirillum  cholera?  asiaticce;  Microspira  comma; 
Vibrio  cholerce. 

Disease  Produced. — Asiatic  cholera  in  man. 

Koch,  in  1883,  discovered  the  specific  cause  of  Asiatic  cholera 
in  the  rice-water  stools  of  cholera  patients. 

Distribution. — The  disease  is  endemic  in  India  and  possibly 

24 


370  VETERINARY    BACTERIOLOGY 

parts  of  China.  It  has  swept  in  epidemics  over  Europe  several 
times  within  the  last  century. 

Morphology  and  Staining. — The  cholera  spirillum  is  a  short, 
slightly  curved  rod,  whence  the  common  designation  of  "  comma 
bacillus."  Longer  filaments  and  involution  forms  are  frequently 
observed.  It  is  motile  by  means  of  a  single  polar  flagellum. 
Spores  and  capsules  are  not  produced.  It  stains  readily  with 
the  common  anilin  dyes  and  is  gram-negative. 

Isolation  and  Culture. — It  may  be  readily  isolated  from  the 
stools  of  cholera  patients  by  plating  upon  nutrient  gelatin.  The 
cultural  characters  are  very  similar  to  those  of  Sp.  metchnikovi. 


., 

.'•*«.     »• 

:         «      *-- 

'  —  < 
- 

-%  ^ 

• 


-   >v 


o  <•  . 


Fig.  158.  —  Spirillum  cholera  (Kiihne-       Fig.  159.—  Spirillum  cholera,  showing 
maim).  flagella  (Giinther). 

Physiology.  —  It  is  an  aerobic  organism.  Growth  occurs  readily 
at  room-temperatures,  although  the  optimum  is  blood-heat.  The 
thermal  death-point  is  60°.  Desiccation,  disinfectants,  and  sun- 
light quickly  destroy  it.  Blood-serum  and  gelatin  are  liquefied. 
Milk  is  not  coagulated.  Nitrates  are  reduced  to  nitrites.  Indol 
is  produced.  The  organism  requires  a  neutral  or  slightly  alkaline 
medium  for  its  development. 

Pathogenesis.  —  Experimental  Evidence.  —  Asiatic  cholera,  in  the 
form  found  in  man,  usually  cannot  be  transmitted  to  laboratory 
animals.  The  etiologic  relation  of  Sp.  choleras  to  the  disease,  how- 
ever, has  been  established  by  accidental  and  intentional  infection 
of  several  laboratory  workers.  Intraperitoneal  injection  of  the 


VIBRIO   OR   CHOLERA    SPIRILLUM   GROUP  371 

guinea-pig  results  fatally.  Ingest  ion  of  the  organisms  by  young 
rabbits  may  result  in  the  development  of  the  symptoms  and  lesions 
of  typical  cholera  as  they  are  observed  in  man. 

Character  of  Disease  and  Lesions  in  Man. — Asiatic  cholera  is 
characterized  by  a  severe  diarrhea.  The  intestinal  epithelium 
is  desquamated,  and  gives  rise  to  the  appearance  known  as  "  rice- 
water  "  stools.  The  intestinal  tract  shows  congestion  and  some- 
times extensive,  even  diphtheritic,  necrosis.  The  loss  of  water 
from  the  blood  results  in  a  considerable,  frequently  fatal,  diminu- 
tion in  blood-pressure. 

Immunity. — True  toxins  have  been  demonstrated  for  the 
cholera  spirillum.  They  are  produced  under  peculiar  cultural 
conditions.  Antitoxin  has  also  been  produced.  Agglutinins  and 
precipitins  are  found  in  immune  blood,  and  frequently  in  the  blood 
of  patients.  Bacteriolysins  may  be  readily  demonstrated;  in 
fact,  it  is  with  this  organism  that  the  classic  demonstration  of 
Pfeiffer's  phenomenon  was  carried  out. 

Active  immunization  against  Asiatic  cholera  has  been  exten- 
sively practised.  The  vaccine  consists  either  of  cultures  killed 
at  58°  or  of  cultures  attenuated  by  growth  at  39°. 

Bacteriological  Diagnosis. — This  may  be  accomplished  by 
an  examination  of  a  stained  mount  from  the  rice-water  discharges, 
by  isolation  of  the  organism,  or  by  the  agglutination  test. 

Transmission. — The  disease  is  transmitted  through  impure 
water  and  food,  particularly  vegetables,  contaminated  with  the 
excretions  of  cholera  patients. 

Non-pathogenic  Spirilla 

Many  species  of  spirilla  closely  resembling  the  preceding  in 
morphology  and  cultural  characters,  but  lacking  in  pathogenicity, 
have  been  isolated  from  a  variety  of  sources.  Such  are  the 
Vibrio  proteus  or  Spirillum  of  Finkler  and  Prior,  isolated  from  the 
stools  in  a  case  of  cholera  nostras,  Spirillum  tyrogenum,  or  Deneke's 
spirillum  from  cheese,  Spirillum  phosphorescens,  isolated  from 
water,  and  many  others. 


CHAPTER  XXXVI 

ACTINOMYCES  GROUP 

THE  members  of  this  group  are  often  called  Trichomycetes 
or  thread  fungi.  In  many  of  their  morphological  characters 
they  resemble  bacteria.  Frequently  they  occur  as  short  rods  that 
cannot  be  differentiated  by  examination  from  true  bacilli.  Usually, 
however,  they  occur  in  threads,  which  in  some  genera  may  be 
branched.  These  threads  may  show  more  or  less  differentiation 
into  parts,  and  certain  portions  may  develop  into  conidia  or  spores. 
These  organisms  show  a  more  complex  life  history,  therefore,  than 
do  the  true  bacteria.  On  the  other  hand,  they  can  scarcely  be 
grouped  with  the  true  molds,  as  they  are  much  simpler  in  structure. 
They  may  be  considered  as  a  group,  therefore,  related  closely  to 
both  bacteria  and  molds,  and  partaking  of  the  nature  of  each. 

These  organisms  show  such  diversity  of  morphology  in  the 
animal  body  and  in  culture-media  that  a  satisfactory  classifica- 
tion into  species  and  genera  is  a  difficult  problem.  Many  generic 
names  have  been  proposed.  Some  of  these  are  valid,  but  the 
organisms  belonging  to  them  are  non-pathogenic,  so  far  as  known. 
Jordan,  in  his  General  Bacteriology,  has  given  a  fairly  satisfactory 
working  classification  for  the  genera  of  the  trichomycetes. 

Filaments  showing  no  branching Lcjtlot/iri.r. 

Filaments  showing  false  branching ('ludothr/.r. 

Filaments  showing  true  branching: 

Spores  or  conidia  produced Noc<ir<lin 

No  spores  demonstrated  Actinomyces 

The  genus  name  Streptothrix  is  also  frequently  used  for  the 
genera  given  above  as  Nocardia  and  Actinomyces.  This  name, 
however,  was  applied  to  an  entirely  different  genus  of  plants  by 
C'onla  in  1839,  and  was  first  so  used.  Its  retention  as  a  genus 
name  in  this  group  is,  therefore,  no  longer  tenable,  in  the  opinion 
373 


ACTIXOMYCES   GROUP  373 

of  many  investigators,  although  a  committee  of  the  Pathological 
Society  has  decided  that  it  be  retained. 

Organisms  belonging  to  Leptothrix  and  Cladothrix  are  not 
known  to  produce  disease  in  animals.  It  seems  to  the  writer  that, 
at  the  present  time,  an  attempt  to  draw  a  distinction  between 
Nocardia  and  Actinomyces  is  impracticable.  The  genera  are  so 
imperfectly  known,  and  their  life-history  in  many  cases  so  imper- 
fectly worked  out,  that  it  is  difficult  to  know  into  which  of  the 
two  genera  to  place  a  particular  organism.  The  genus  name 
Actinomyces  will  be  here  used  to  include  both. 


Fig.  160. — Streptothrix  (Actinomyces)  ccelicolor,  a  non-pathogenic  tricho- 
mycete  from  the  soil.  A  ring  colony  on  a  semisolid  medium  showing  filaments 
and  aerial  hyphae  (Miiller). 

Several  organisms  are  included  in  this  genus,  as  here  dis- 
cussed, that  may  be  shown  to  belong  to  the  bacilli  and  not  to  the 
trichomycetes. 

Many  species  of  organisms  of  this  group  are  known  from  the 
descriptions  of  a  single  author  only.  It  is  difficult  to  determine 
from  these  descriptions  how  many  are  valid  species  and  how  many 
merely  synonyms.  The  facts  seem  to  be  that  Actinomyces  are 
widely  distributed  in  nature.  They  may  be  isolated  in  abundance 


374  VETERINARY   BACTERIOLOGY 

from  most  soils,  and  may  be  found  to  develop  upon  almost  any 
plate  of  medium  exposed  to  the  air.  Only  under  exceptional 
conditions  are  they  pathogenic,  but  it  is  probable  that  the  species 
usually  described  as  such  are  normally  saprophytes  that  can, 
upon  occasion,  proliferate  in  the  tissues  of  the  body  and  produce 
disease.  The  fact  that  cattle  become  infected  through  the  gums 
or  tongue,  where  the  awns  of  certain  grasses  penetrate,  that  the 
barley  testers,  who  bite  the  barley  grain  to  determine  its  brewing 
quality,  are  most  frequently  infected  among  men  in  temperate 
climates,  that  injuries  to  the  feet  of  natives  of  certain  tropical 
countries  (where  no  adequate  protection  is  worn  on  the  feet)  are 


Fig.  161. — Streptothrix  (Actinomyces)  coelicolor.    Colony  on  agar.    This  colony 
structure  is  quite  typical  of  many  species  (Muller). 

frequently  followed  by  local  infections,  and  that  Actinomyces  have 
been  found  causing  infections  in  practically  all  domestic  animals, 
by  one  investigator  or  another  is  evidence  of  the  wide  distribution 
of  the  members  of  the  genus.  The  species  to  be  described  are 
Actinomyces  bovis  and  A.  nocardii  in  cattle,  A.  caprcr  in  goats, 
A.  madurce,  and  A.  eppingeri  in  man. 

The  group,  as  a  whole,  may  be  characterized  as  consisting  of 
slender,  branching  organisms,  which  may  develop  into  colonies 
made  up  not  only  of  threads  but  rods,  cocci,  and  other  cell  forms. 
Frequently,  in  animal  ti^m-s  :m<l  sometimes  upon  artificial  incd'ui, 
the  ends  of  the  threads  nuiy  l*e  dubbed.  When  grown  upon  the 
surface  of  artificial  media  sonic  forms  develop  aerial  hyphae, 


ACTINOMYCES   GROUP  375 

which  segment  into  chains  of  conidia.  All  species  retain  the  Gram 
stain  to  a  greater  or  less  degree.  Some  are  aerobic,  others  facul- 
tative, and  still  others  obligate  anaerobes.  Pigments  are  produced 
by  some  species. 

Actinomyces  bovis 

Synonyms. — Streptothrix  bovis;  Cladothrix  actinomyces;  Strepto- 
thrix  actinomyces;  Discomyces  boms. 

Disease  Produced. — Lumpy  jaw  and  wooden  tongue  (actino- 
mycosis)  in  cattle,  and  probably  related  infections  in  other  animals 
and  man. 

Harz,  in  1878,  gave  the  name  Actinomyces  to  the  ray-fungus, 
which  Bellinger,  in  the  preceding  year,  had  found  present  in  the 
characteristic  tumor-like  growths  in  cattle. 

Distribution. — The  infection  is  known  from  Europe  and  North 
and  South  America. 

Morphology  and  Staining. — In  the  infected  tissues  the  organism 
forms  minute  yellowish  granules,  sometimes  large  enough  to  be 
readily  observed  by  the  unaided  eye.  These  granules  are  made  up 
of  compact  masses  of  the  organisms.  Branched  filaments,  with  a 
more  or  less  radial  arrangement,  are  to  be  observed  occupying 
the  central  portion,  commonly  mixed  with  coccus-like  degeneration 
products.  The  margin  of  the  granule  or  rosette,  when  examined 
in  cross-section,  is  found  to  consist  of  club-like  enlargements  of 
the  threads,  showing  a  marked  refractivity  to  light.  The  filaments 
are  slender,  usually  about  0.5  /w  in  diameter.  It  is  believed  that 
the  formation  of  the  clubbed  ends  is  correlated  in  some  way  with 
the  resistance  of  tissue  to  invasion.  They  have  been  variously 
regarded  as  degeneration  products,  involution  forms,  and  as  indi- 
cating a  thickening  of  the  sheath  to  protect  the  organism  against 
antibodies  produced  by  the  tissues.  Young  colonies  on  artificial 
media  consist  of  interlacing,  branched  threads,  which  tend  to  form 
compact  masses.  These  commonly  break  up  into  bacillus-like 
segments,  in  a  manner  not  unlike  the  formation  of  certain  spores 
among  higher  fungi,  by  segmentation  of  the  hyphal  threads. 
Whether  or  not  these  correspond  to  the  oi'dial  type  of  spore  pro- 
duced in  the  higher  fungi,  or  represent  spores  at  all,  is  not  known. 
The  clubbed  type  rarely  develops  in  artificial  media.  The  organism 


376  VETERINARY   BACTERIOLOGY 

stains  readily  with  the  common  anilin  dyes  and  is  gram-positive. 
It  is  not  acid-fast. 

Isolation  and  Culture. — The  organism  is  not  easily  isolated 
in  pure  cultures,  particularly  when  it  occurs  in  mixed  cultures 
with  pyogenic  cocci  in  the  lesions.  Wright  has  described  a  technic 
which  he  found  quite  uniformly  successful.  Pus  or  tissues  contain- 
ing the  organism  in  filamentous  rosettes  is  preferable  to  that  con- 
taining only  the  clubbed  type,  as  in  the  latter  degeneration  has 
gone  so  far  that  frequently  no  growth  will  occur.  The  granules 
are  washed  in  sterile  water,  crushed  between  sterile  slides,  and 
inoculated  in  varying  amounts  into  tubes  of  melted  1  per  cent, 
dextrose  agar,  and  incubated  at  37°.  In  his  experience  the 

colonies  developed  character- 
istically  from    5    to    12   mm. 

jritfH 

below  the  surface,  but  others 
have  found  them  to  form 
G&  quite  as  well  upon  the  surface 
of  the  medium.  Isolated  col- 
onies may  then  be  transferred 
to  other  media.  In  bouillon 
the  organism  forms  distinct. 
solid,  spherical,  or  mulberry- 
like  masses  at  the  bottom  of 
the  tube.  Growth  is  secured 

Fig.  m.—Actinomycc8  bovis,  tissue      with   difficulty  upon   the  sur- 
section  showing  the  radial  arrangement       „  ..    t  , . 

and  the  clubbing  of  threads  (Gunther).  face  of  the  medmm,  accord- 
ing to  Wright,  but  other  in- 
vestigators have  not  experienced  the  same  difficulty.  It  forms 
on  agar  and  glycerin  agar  colonies,  which  at  first  resemble  tiny 
drops  of  amber;  these  enlarge,  and  either  remain  discrete  or 
coalesce  to  form  a  distinctly  wrinkled,  "  lichen-like "  membrane, 
which  frequently  has  a  dusty  appearance.  Gelatin  is  slowly 
liquefied. 

Physiology. — The  organism  may  be  regarded  as  a  facultative 
aerobe,  as  growth  appears  to  take  place  best  under  anaerobic 
conditions.  The  optimum  growth  temperature  is  37°.  The 
organism  is  resistant  to  desiccation  and  will  live  for  a  long  period, 
probably  months,  in  a  dried  condition.  Gelatin  is  liquefied. 


ACTINOMYCES   GROUP 


377 


There  is  no  gas-  or  acid-production.     A  brown  to  black  pigment 
may  be  produced. 

Pathogenesis. — Experimental  Evidence. — In  the  great  majority 
of  cases  experimental  inoculation  is  without  result.     Many  animals 
have  been  used — cattle,  sheep,  swine,  dogs,  cats,  rabbits,  and 
guinea-pigs.     In  relatively  a  few  cases  significant  lesions  have 
been    developed.     Musgrave,    Clegg,    and    Polk    have   produced 
extensive  suppurative  lesions  by  intraperitoneal  inoculation  of  the 
monkey,  the  infection  terminating  fatally  in  about  three  weeks. 
The  common  lack  of  pathogenesis  may  be  due  to  differences  in 
resistance,  to  a  diminution 
of  virulence  due  to  cultiva- 
tion, or  to  the  manner  of 
inoculation.      In    cattle    it 
may  gain  entrance  with  a 
grass   awn,    and    this   may 
protect    it    from    the    de- 
structive   agencies    of    the 
tissues  until   its  pathogen- 
icity  is  well  established. 

Character  of  Disease  and 
Lesions  Produced. — A  swell- 
ing or  tumor-like  mass  de- 
velops in  cattle  at  the  site 
of  infection.  This  softens 
and  ultimately  discharges 
thick,  yellowish  pus.  The 
discharge  after  the  lesion  has  opened  may  become  intermittent 
in  character.  When  the  tongue  is  the  primary  seat  of  infection 
it  becomes  swollen,  indurated,  and  protrudes  from  the  mouth  in 
some  cases.  The  bones  of  the  jaw  are  often  attacked.  The 
infection  is  chronic.  Animals  rarely  die  from  immediate  effects. 
In  a  few  instances  metastatic  infection  of  other  parts  of  the  body 
than  the  head  and  neck  have  been  reported. 

In  man  the  disease  usually  attacks  the  softer  tissues,  progresses 
more  rapidly  than  in  cattle,  and  is  apt  to  terminate  fatally  from 
metastatic  infection.  Whether  or  not  the  organism  isolated  from 
human  actinomycosis  is  the  same  as  that  found  in  cattle  is  uncer- 


Fig.  163. — Actinomyces  bovis  (strepto- 
thrix  actinomyces),  stained  mount  from  cul- 
ture-medium (Musgrave,  Clegg,  and  Polk, 
in  "  Philippine  Journal  of  Science  "). 


378  VETERINARY    BACTERIOLOGY 

tain.  The  same  may  be  said  of  the  forms  that  have  been  isolated 
from  similar  infections  in  other  animals,  among  them  the  horse, 
dog,  and  pig. 

Immunity. — No  method  of  immunization  against  the  disease 
has  been  developed. 

Bacteriological  Diagnosis. — A  microscopic  examination  of  the 
unstained  pus  will  usually  reveal  the  characteristic  granules, 
with  the  radial  arrangement  of  clubs  or  of  tangled  bits  of  branched 
threads.  A  film  stained  by  Gram's  method  will  bring  the  latter 
out  clearly  when  present  in  small  numbers  only. 

Transmission. — It  is  believed  that  the  organism  commonly 
enters  the  body  through  a  trauma,  through  carious  teeth,  or  by 
being  carried  into  the  tongue  or  the  gum  with  the  sharp  awns  of 
certain  grasses  and  grains.  So  far  as  known  the  disease  is  wholly 
non-contagious. 

Actinomyces  nocardii 

Synonyms. — Streptothrix  nocardii;  Actinomyces  farcinica;  Strep- 
tothrix  farcinica;  Nocardia  farcinica. 

Disease  Produced. — Bovine  farcy.     Farcin  du  boeuf. 

Nocard,  in  1888,  first  described  an  Actinomyces  or  Strepto- 
thrix from  the  lesions  of  cattle  in  Guadeloupe  suffering  from  a 
disease  termed  bovine  farcy.  The  disease  itself  has  not  been 
adequately  studied,  although  the  organism  has  been  investigated 
by  several  workers.  There  is  no  record  of  its  occurrence  in  the 
United  States. 

Morphology  and  Staining. — The  organism  is  slender,  much 
branched,  and  interwoven.  In  culture-media  short,  plump  fila- 
ments with  branches  may  occur,  and  in  old  cultures  many  ovoid 
cells  are  found.  The  organism  is  gram-positive,  and  many  por- 
tions, particularly  in  old  cultures,  are  acid-fast  and  also  alcohol- 
fast. 

Isolation  and  Culture. — It  may  be  isolated  in  pure  culture  from 
lesions  directly  upon  artificial  media.  The  colonies  upon  agar 
are  small,  white,  irregular,  raised,  and  opaque.  Upon  glycerin 
agar  they  are  at  first  discrete,  but  soon  coalesce,  and  present 
a  moist,  meal-like  growth.  Bouillon  is  never  clouded,  but  a 
grayish,  flocculent  mass  forms  at  the  bottom.  Milk  is  unchanged. 

Physiology. — The  organism    is   a   facultative   ae'robe.     It  de- 


ACTINOMYCES  GROUP  379 

velops  best  at  37°.     It  is  resistant  to  desiccation,  and  maintains 
its  virulence  when  cultivated. 

Pathogenesis. — Experimental  Evidence. — Guinea-pigs  are  easily 
infected  by  intraperitoneal  injections.  The  organism  produces 
numerous  nodules  resembling  tubercles  upon  the  peritoneum 
and  the  abdominal  organs,  particularly  the  liver,  spleen,  and 
kidneys.  Intravenous  injection  gives  rise  to  a  condition  resembling 
generalized  miliary  tuberculosis.  Intraperitoneal  injection  of  the 
monkey  gives  rise  to  similar  lesions.  Cattle  and  sheep  develop, 
at  the  point  of  a  subcutaneous  inoculation,  an  abscess  which  dis- 


Fig.    164. — Actinomyces  nocardii,   stained   mount    from   culture    (Musgrave, 
Clegg,  and  Polk,  in  "Philippine  Journal  of  Science"). 

charges,  ulcerates,  and  may  disappear,  to  reappear  after  an 
interval. 

Character  of  Disease  and  Lesions. — The  disease  in  cattle  is 
characterized  by  an  enlargement  of  the  superficial  lymph-nodes, 
which  ulcerate  and  have  much  the  appearance  of  farcy  in  the  horse. 
The  internal  organs  may  be  affected,  with  a  resultant  pseudo- 
tuberculosis. 

Immunity. — Methods  of  immunization  have  not  been  developed. 

Bacteriological  Diagnosis. — The  organism  may  be  recognized 
in  preparations  from  the  lesions,  but,  for  differentiation  from  other 


380  VETERINARY  BACTERIOLOGY 

Actinomyces  or   Streptothrices,  culture  and  animal  inoculation 
are  necessary. 

Transmission. — The  disease  is  probably  transmitted  by  wound 
infection,  but  this  is  not  certainly  known. 

Actinomyces  caprae 

Synonyms. — Streptothrix  caprce  and  possibly  S.  cam's. 

Disease  Produced. — Actinomycosis  (streptothricosis)  in  goats, 
possibly  in  the  dog. 

Silberschmidt,  in  1899,  published  a  description  of  an  organism 
belonging  to  this  group,  which  he  isolated  from  a  goat  affected 


"'"    '•  '"v 

wrv\ 


Fig.  165. — Actinomyces  caprce,  stained  mount  from  culture  (Musgrave,  Clegg, 
and  Polk,  in  "Philippine  Journal  of  Science"). 

with  a  disease  which  closely  simulated  tuberculosis.  It  has  been 
studied  by  several  other  investigators,  who  are  in  agreement  that 
it  should  be  regarded  as  a  distinct  species. 

Morphology  and  Staining. — Morphologically,  it  resembles  the 
true  bacteria  more  than  other  members  of  this  group.  The  fila- 
ments are  comparatively  short,  and  show  little  tendency  to  form 
tangled  masses,  but  separate  easily.  Both  in  culture  and  in  the 
lesions  rod  forms  and  cocci  are  predominant.  It  stains  with  the 
anilin  dyes  rather  irregularly,  and  is  alcohol  and  acid-fast. 

Isolation  and  Culture. — The  organism  grows  rather  readily 
upon  most  of  the  laboratory  media,  so  that  isolation  is  not  a  matter 


ACTINOMYCES   GROUP  381 

of  difficulty.  Upon  agar  the  growth  appears  in  two  to  three  days 
as  small,  brownish  colonies.  It  is  somewhat  more  luxuriant  upon 
glycerin  and  maltose  agar,  the  colonies  coalescing  to  give  the 
growth  a  moist,  mealy  appearance.  The  colonies  are  light  brown 
in  color.  Growth  upon  potato  is  similar.  In  bouillon  the  colonies 
develop  upon  the  surface  as  fine  dry  disks,  and  form  a  pellicle, 
which  finally  settles  as  a  sediment,  the  broth  remaining  clear. 

Physiology. — The  organism  is  a  facultative  ae'robe. 

Pathogenesis. — The  organism  produces  tubercle-like  lesions 
in  the  rabbit,  guinea-pig,  and  monkey  upon  inoculation.  It  is  not 
of  any  considerable  economic  importance. 

Actinomyces  madurae 

Synonym. — Streptothrix  madurce. 

Disease  Produced. — Madura  foot,  mycetoma,  streptothricosis 
in  man. 

Vincent,  in  1894,  cultivated  an  Actinomyces  from  cases  of 
mycetoma  or  Madura  foot  in  man.  This  disease  occurs  in  certain 


Fig.  166. — Actinomyces  madurce,  stained  mount  from  culture  (Musgrave,  Clegg, 
and  Polk,  in  "  Philippine  Journal  of  Science  "). 

tropical  countries,  as  southern  Asia  and  the  Philippines.  It  is 
undoubtedly  a  different  species  from  those  already  described.  It 
is  not  known  to  affect  animals  in  nature,  but  will  infect  the  monkey 
upon  intraperitoneal  inoculation. 


382  VETERINARY   BACTERIOLOGY 

Actinomyces  eppingcri 

Synonyms. — Streptothrix  eppingeri;  Streptothrix  freeri. 

Disease  Produced. — Mycetoma,  or  Madura  foot,  and  other 
lesions  in  man. 

This  organism  was  described  by  Eppinger,  and  has  since  that 
time  been  found  in  various  actinomycotic  infections  in  man. 
It  is  pathogenic  for  the  monkey,  guinea-pigs,  and  rabbits,  but  is 
not  known  to  produce  disease  in  animals  under  natural  conditions. 

ACTINOMYCES  OF  OTHER  INFECTIONS 

Probably  about  thirty  or  more  other  species  have  been  described 
belonging  to  this  genus.  In  most  cases  they  have  been  reported 
but  once  or  have  been  incompletely  described.  As  has  before 
been  emphasized,  careful  work  is  still  needed  in  order  to  deter- 
mine the  true  number  of  valid  species  and  their  relationship  to 
disease. 


CHAPTER   XXXVII 


BLASTOMYCETES 

THK  genus  name  Blastomyces  is  used  to  designate  a  group  of 
pathogenic  fungi  having  many  points  in  common  with  the  members 
of  the  genera  Saccharomyces  and  possibly  Torula.  It  is  not 
certainly  known  that  the  forms  thus  classified  are  closely  related 
among  themselves,  for  it  is  a  well-known  fact  that  many  of  the 
Hyphomycetes,  when  grown  in  certain  culture-media,  will  assume 
a  form  indistinguishable  from  the  yeasts.  It  is  possible,  therefore, 
that  some  of  the  forms  described  as  members  of  the  genus  Blasto- 
myces may  be  only  growth 
stages  of  higher  forms.  Here, 
again,  as  has  been  emphasized 
in  other  groups,  there  is  need 
still  for  careful  morphological 
and  cultural  studies  of  the 
various  species  that  have  been 
described,  for  some  of  them  are 
very  imperfectly  known. 

An  understanding  of  the 
morphology  of  the  Blastomyces 
can  best  be  obtained  by  a  pre- 
liminary discussion  of  the  Sac- 
charomyces or  true  yeasts. 
The  one  character  which  sep- 
arates this  genus  from  the  Hyphomycetes  is  the  difference  in  the 
vegetative  method  of  reproduction.  This  is  accomplished  by 
budding.  The  mother-cell  is  usually  oval  or  round,  and,  at 
various  points  on  its  surface,  produces  small  buds,  which  enlarge 
and  soon  separate  as  independent  cells.  Occasionally  these  cells 
may  remain  together  and  become  considerably  elongated.  By 
continued  budding  from  the  tip,  a  chain  of  cells  is  formed 

383 


Fig.    167.  —  Brewer's    yeast,    Snccha- 
ronnjces  cerevisioe  (Glint  her). 


384  VETERINARY   BACTERIOLOGY 

simulating  a  mycelial  thread  of  one  of  the  Hyphomycetes.  The 
cell  differs  from  that  of  a  bacterium  by  the  presence  of  a  definite 
nucleus,  which  may  be  demonstrated  by  careful  staining  technic. 

Spores  are  produced  by  some  yeasts  when  the  cells  are  brought 
under  the  right  conditions  of  moisture,  oxygen  pressure,  and 
temperature.  Generally,  two,  four,  or  six  are  produced  within 
a  single  cell.  This  type  of  spore  formation  relates  such  forms 
definitely  to  the  higher  fungi,  known  as  Ascomycetes  or  sac 
fungi.  In  these  fungi  the  spores  are  borne  in  sacs  or  asci  (singular, 
ascus),  and  the  cell  of  the  yeast,  with  its  contained  spores,  is  sup- 
posed to  represent  a  simple  type  of  ascus.  Resting  cells,  consist- 
ing of  heavily  walled  or  encapsulated  cells  filled  with  protein, 
glycogen,  or  oil-granules,  are  formed  by  many  yeasts.  These 
granules  may  resemble  spores,  and  have  doubtless  many  times  been 
mistaken  for  them.  When  brought  under  favorable  conditions 
the  cell,  as  a  whole,  begins  again  to  produce  buds,  showing  con- 
clusively that  the  granules  cannot  be  regarded  as  spores. 

Among  the  true  yeasts,  those  which  are  not  known  to  produce 
spores  are  sometimes  placed  in  the  form  genus  Torula.  It  is  not  cus- 
tomary to  make  this  distinction  among  the  pathogenic  yeasts  or 
Blastomyces,  although  it  has  been  attempted  by  some  authors. 
As  here  used,  the  term  Blastomyces  includes  all  those  pathogenic 
forms  which  reproduce  regularly  by  budding,  and  may  or  may  not 
produce  ascospores. 

The  organisms  belonging  to  this  group  are  Blastomyces  farci- 
iti/nosus,  B.  dermatitidis,  and  B.  coccidioides. 

Blastomyces  farciminosus 

Synonyms. — Cryptococcus  farciminosus;  Leishmania  farci- 
minosa. 

Disease  Produced. — Blastomycotic  epizootic  lymphangitis  or 
peeudofarcy  in  the  horse. 

Rivolta,  in  1873,  first  described  the  organism  associated  with 
this  disease.  Tokoshige,  in  1897,  cultivated  the  organism  and 
determined  its  classification.  It  has  been  studied  since  that  time 
by  several  investigators.  Galli-Valerio  contends  that  this  organ- 
ism is  a  protozoan  and  not  a  Blastomyces.  There  is  a  clinically 
similar  disease,  since  described  in  Europe  and  the  United  States 


BLASTOMYCETES  385 

as  due  to  a  member  of  the  mold  genus  Sporotrichum.  The  organ- 
ism described  by  Tokoshige  should  be  reinvestigated.  It  is  pos- 
sible that  it  may  prove  to  be  a  Sporotrichum  also. 

Distribution.  —  The  disease  is  known  from  Italy,  Egypt,  Tunis, 
England,  France,  northern  Europe,  Japan,  India,  the  Philippines, 
and  possibly  the  United  States  (North  Dakota,  Iowa). 

Morphology  and  Staining.  —  The  organism  as  it  occurs  in  tissues 
does  not  show  budding  forms  ordinarily,  but  reproduces  by  a 
series  of  sporulations.  In  iriounts  prepared  from  the  tissues  it 
has  a  double  refractive  contour,  which  makes  it  stand  out  dis- 
tinctly from  the  remainder,  even  when  unstained.  It  is  usually 
spherical  or  ovoid,  3  to  4  ,u  in  diameter.  The  cell  contents  may  be 
homogeneous  or  granular.  In  culture-media  the  organism  consists 
of  hyphal  and  spherical  forms.  Cells 
with  buds  may  be  found,  identifying 
the  organism  definitely  with  the  Blas- 
tomycetes.  Cells  containing  granules, 
and  resembling  closely  the  resting  cells 
of  the  yeasts,  are  common.  It  has 
not  been  conclusively  shown  that  true 
sporulation  takes  place  in  culture- 
media.  The  organism  stains  readily  Fig.  168.—  Blastomyces  far- 


with  aqueous  anilin  dyes  and  is  gram-     «»»™™;  cells  from  culture- 

(adapted  from  Toko- 


positive.     The  latter  method  of  stain-     shige). 

ing  is  useful  in  demonstrating  the  or- 

ganism in  pus  or  tissues.     The  alcohol  must  not  remain  too  long 

in  contact  with  the  organism  or  it  will  lose  color. 

Isolation  and  Culture.  —  The  R.  fardminosus  is  isolated  upon 
culture-media  with  considerable  difficulty.  Several  investigators, 
particularly  those  holding  to  the  protozoan  nature  of  the  organism, 
deny  that  it  has  been  accomplished.  In  view  of  the  success  which 
has  attended  the  cultivation  of  an  essentially  similar  organism 
in  man,  there  seems  no  good  reason  to  deny  that  Tokoshige  and 
others  have  succeeded  in  securing  it  in  pure  cultures.  A  slightly 
acid  medium  is  said  to  be  more  favorable  than  one  which  is  alkaline. 
Growth  is  very  slow  in  any  event. 

Bouillon  finally  shows  a  white,  flocculent  deposit.  Upon 
the  surface  of  agar,  gray-white  granular  colonies  make  their  ap- 


386  VETERINARY   BACTERIOLOGY 

pearance  in  the  course  of  a  month,  and  finally  attain  to  a  diameter 
of  1  to  4  mm.  The  colony  is  wrinkled,  and  can  be  removed  only 
with  difficulty.  Growth  upon  gelatin  is  essentially  similar. 
Potato  seems  somewhat  more  favorable,  and  growth  occurs  more1 
rapidly,  but  is  of  the  same  character  as  on  agar. 

Physiology. — The  organism  is  aerobic.  Growth  occurs  at 
room-temperature  as  well  as  at  37°.  It  does  not  liquefy  gelatin. 
Sugars  are  not  fermented  with  production  of  either  acid  or  gas. 

Pathogenesis. — Experimental  Evidence. — Guinea-pigs  and  rab- 
bits are  not  easily  infected  with  pure  cultures.  Typical  lesions 
have  been  produced  in  the  horse  by  Tokoshige.  They  are  readily 
produced  by  the  injection  of  pus  from  natural  infections. 

Character  of  Disease  and  Lesions. — The  disease  in  the  horse 
shows  a  marked  superficial  resemblance  to  farcy.  The  infection 
progresses  through  the  subcutaneous  lymphatics  and  forms  dis- 
tinct nodules.  These  may  suppurate.  Metastatic  infection  of 
the  internal  organs  occasionally  occurs. 

Immunity. — No  practicable  method  of  immunization  has  been 
developed. 

Bacteriological  Diagnosis. — The  organism  may  be  readily 
observed  in  a  mount  of  the  pus  from  a  lesion  stained  by  Gram's 
method. 

Transmission. — It  is  supposed  that  infection  is  traumatic,  that 
the  organism  gains  entrance  through  cutaneous  lesions.  The 
disease  not  highly  contagious. 

Blastomyccs  dermatitidis 

Synonyms. — Saccharomyces  dermatitidis',  Oldium  dermatitidis. 

Disease. — Blastomycetic  dermatitis  in  man. 

Busse,  in  1894,  first  described  an  organism  of  this  group  as  the 
cause  of  a  fatal  infection  in  man.  Gilchrist,  in  1896,  found  a 
similar  organism  as  the  cause  of  a  dermatitis  in  man.  Since  that 
time  the  organism  has  been  repeatedly  isolated  and  studied. 

Distribution. — Blastomycotic  dermatitis  has  been  reported 
from  the  United  States,  the  Philippines,  and  Europe. 

Morphology  and  Staining. — It  is  probable  that  several  distinct 
species  have  been  grouped  together;  that  is,  not  all  cases  have 
shown  morphologically  identical  organisms  to  be  present.  They 


BLASTOMYCETES  387 

have  not  as  yet  been  sufficiently  studied  to  justify  their  separation 
as  distinct  species,  but  will  be  treated  rather  as  one  polymorphic 
form.  Careful  morphological  and  cultural  studies  are  still  needed. 
In  the  tissues  the  organisms  appear  almost  invariably  as 
budding  forms.  The  cells  are  spherical  or  ovoid,  from  10  to  17  (J> 
in  diameter.  They  are  distinctly  double  contoured.  Several 
investigators  have  observed  what  they  believe  to  be  sporulating 
forms.  The  cells  are  frequently  granular  or  vacuolate,  resembling 
typical  yeast  cells  in  this  respect.  Upon  culture-media  numerous 
hyphal  threads  and  budding  cells  are  produced. 


'1'f-^' 


Fig.    169. — Blastomyces  dermatitidis.     Budding  forms  and  mycelial   growth 
from  glucose  agar  (Irons  and  Graham,  in  "Journal  of  Infectious  Diseases"). 

The  organisms  do  not  stain  very  readily  with  the  aqueous 
anilin  dyes. 

Isolation  and  Culture. — Isolation  of  the  organism  is  usually 
attended  with  considerable  difficulty.  Blood-serum  slants  are 
usually  employed  and  inoculated  with  material  from  the  lesion. 
Repeated  trials  are  sometimes  necessary  before  a  growth  is  secured. 
After  once  accustomed  to  growth  on  artificial  media,  no  difficulty 
is  found  in  getting  the  organism  to  develop  upon  most  of  the 
common  culture-media. 

Small  white  colonies  showing  a  mold-like  surface,  due  to  the 
formation  of  numerous  aerial  hyphae,  develop  upon  the  surface 


388 


VETERINARY   BACTERIOLOGY 


of  agar.  The  addition  of  dextrose  to  the  medium  somewhat  in- 
creases the  luxuriance  of  the  growth.  In  bouillon  a  fluffy,  mold- 
like  colony  or  a  granular  sediment  develops  without  any  evidence 
of  the  diffuse  clouding  generally  found  in  yeast  cultures.  Gelatin 
is  not  liquefied.  Milk  may  or  may  not  show  coagulation  and 
slight  digestion  of  the  casein.  Potato  is  a  favorable  medium. 

Physiology. — Growth  occurs  at  room-temperatures,  but  some- 
what more  luxuriantly  at  37°.     The  organism  is  aerobic  and  facul- 


Fig.   170. — Blastomyces  dermatitidis   (Hamburger,  in  "  Journal  of   Infectious 

Diseases"). 

tative  anaerobic.  Gas  and  acids  are  not  produced  in  carbo- 
hydrate media. 

Pathogenesis. — Experimental  Evidence. — Guinea-pigs  and  rab- 
bits may  be  infected,  with  production  of  either  a  local  abscess  or 
generalized  blastomycosis.  The  lesions  resemble  in  their  essential 
characters  those  found  in  the  human  body. 

Character  of  Disease  and  Lesions. — In  man  a  papule  generally 
appears  upon  one  of  the  extremities,  the  face,  or,  more  rarely, 
elsewhere.  A  viscid  pus  is  exuded,  and  there  is  commonly  con- 
siderable enlargement.  Healing  with  an  abundant  formation  of 
cicatricial  tissue  gradually  occurs.  Usually  the  lymphatics  arc 


BLASTOMYCETES  389 

not  involved,  the  disease  differing  in  this  respect  from  the  lymph- 
angitis of  the  horse.  The  course  of  the  disease  is  usually  chronic, 
and  it  may  persist  for  years,  new  ulcers  appearing  successively  on 
various  parts  of  the  body.  Generalization  has  been  reported  in  a 
considerable  number  of  cases.  The  skin  lesions  have  sometimes 
been  confused  with  those  of  syphilis  and  tuberculosis.  Primary 
infection  of  the  lungs  has  been  shown  in  several  cases. 

Immunity. — No  method  of  establishing  immunity  has  been 
developed. 

Bacteriological  Diagnosis. — This  may  be  accomplished  by  direct 
microscopic  examination  of  the  pus.  Phalen  and  Nichols  state 
that  the  organism  may  be  most  easily  demonstrated  by  treating 
unstained  sections  with  potassium  hydrate  and  mounting  in 
glycerin. 

Transmission. — It  is  supposed  that  infection  sometimes  occurs 
through  wounds,  but  several  instances  have  come  to  light  in  which 
the  infection  was  primarily  pulmonary  and  the  skin  lesions  sec- 
ondary. 

Blastomyces  coccidioides 

Synonym. — Oidium  coccidioides. 

Disease  Produced. — Blastomycosis,  so-called  coccidioidal  granu- 
loma  in  man. 

Posades  and  Wernecke,  in  1892,  first  reported  a  case  of  so- 
called  coccidioidal  granuloma  from  Argentina.  In  the  United 
States  the  disease  has  been  recorded  principally  from  California, 
particularly  in  the  San  Joaquin  Valley. 

Morphology. — This  form  is  of  particular  interest,  because  the 
budding  or  true  blast-only ces  form  very  rarely  occurs  in  the  tissues, 
and  multiplication  is  almost  wholly  through  sporulation.  The 
organism  in  the  tissues  is  spherical  and  doubly  contoured.  It  may 
reach  a  diameter  of  30  u  or  even  more.  Budding  forms  have  been 
recorded  from  pus.  In  artificial  media  the  organism  resembles 
a  mold,  but  budding  forms  may  be  observed.  The  method  of 
reproduction  in  tissues,  by  the  formation  of  spores  within  the 
mother-cell  and  their  liberation  by  a  rupture  of  the  cell  membrane, 
has  led  some  investigators  to  believe  that  the  organism  is  roally 
a  protozoan.  However,  sporulation  of  this  general  type  occurs 
in  the  yeasts.  The  question  seems  to  be  definitely  settled  in 


390 


VETERINARY    BACTERIOLOGY 


a.  Typical  large  organism  with 
thick  hyaline  capsule  in  a  giant 
cell. 


6.  Sporulation  from  within  an 
imperfect  giant  cell.  Capsule 
ruptured  in  two  places,  a,  a. 


C2L         _o/i,  £>  & 


c.  Giant  cell  containing  a  num- 
ber of  organisms  without  gram, la 5 
cytoplasm.  These  have  appa- 
rently recently  brrn  lil>cratr<l  l>y 
sporulation  of  their  parent  or- 
ganism. 


Fig.  171.— Blastomyc 


.-.in  ".Iinini:il  of  Infcdioii-  I)i-c;i- 


BLASTOMYCETES  391 

favor  of  the  plant  hypothesis  by  the  culture  forms  on  artificial 
media. 

Pathogenesis. — Whether  this  organism  should  be  distinctly 
separated  from  the  preceding  is  not  certainly  known.  The  disease 
frequently  shows  no  cutaneous  lesions;  the  infection  is  systemic  and 
probably  always  fatal.  There  is  often  an  involvement  of  the 
meninges. 


CHAPTER  XXXVIII 

MOLD  OR  HYPHOMYCETE  GROUP 

THE  term  Hyphomycete,  as  usually  interpreted,  is  one  of  con- 
venience only,  for  within  this  group  are  included  members  of  the 
fourjgreat  divisions  of  fungi  generally  recognized  by  botanists.  The 
members  of  the  group  are  many  times  not  closely  related.  They 
all  resemble  each  other  in  having  a  plant  body  or  mycelium,  which 
consists  of  threads  or  hyphce  made  up,  in  the  majority  of  forms,  of 
chains  of  cells.  Reproduction  is  not  generally  by  budding,1^ 
although  this  may  sometimes  occur.  The  hyphae  themselves  break 
up  into  spores,  or  spores  are  borne  at  the  tips  of  hyphae  that  have 
been  differentiated  for  the  purpose.  The  hyphae  may  unite  to 
form  a  more  or  less  solid  mass,  sometimes  tissue-like  in  appearance. 
This  mass  may  remain  viable  when  dried  for  a  considerable  time, 
and  may  function  in  much  the  same  manner  as  a  resistant  spore 
in  tiding  the  organism  over  unfavorable  conditions.  Such  a 
structure  is  called  a  sclerotium.  The  names  given  to  these  various 
types  of  structures  have  already  been  discussed  under  the  heading 
of  Morphology  in  Section  I.  f^ 

The  Hyphomycetes,  for  the  most  part,  belong  to  the  division 
of  fungi  termed  Fungi  imperfecti  by  the  botanist.  The  name  is 
derived  from  the  fact  that  these  fungi  are  not  known  to  produce 
perfect  or  sexual  spores.  Hundreds  of  genera  and  thousands  of 
species  have  been  described  as  belonging  to  this  group.  Many  of 
these  are  doubtless  simply  developmental  stages  of  forms  that 
are  known  under  other  names.  The  lil<-  history  of  some  fungi 
has  been  found  to  be  so  complex,  and  consists  of  so  many  stages, 
that  five  or  six  names  have  been  applied  and  the  different  stages 
put  in  different  groups  of  fungi,  until  it  was  found  that  all  were 
the  same  polymorphic  species.  Unfortunately,  careful  mor- 
phological study  has  not  been  made  of  the  pathogenic  mcmlx  r^ 
of  this  group,  and  there  is  the  greatest  confusion  in  tho  nonrien- 


MOLD  OR  HYPHOMYCETE  GROUP  393 

« 

clature.  The  pathologists  and  bacteriologists  who  have  de- 
scribed the  organisms  have  rarely  paid  any  attention  to 
their  botanical  relationships,  and  the  organisms  themselves, 
for  the  most  part,  have  been  ignored  by  the  botanist  in  his 
classification. 

As  before  stated,  the  possession  of  a  more  or  less  definite 
mycelium,  a  more  or  less  "  mold-like  "  growth,  and  the  general 
production  of  spores  are  all  that  is  needed  to  include  an  organism 
in  the  group.  Many  of  the  organisms  of  the  group  are  very  com- 
mon in  nature  and  are  pathogenic  only  under  exceptional  condi- 
tions, while  others  have  so  adapted  themselves  to  a  parasitic 
existence  that  they  may  be  regarded  as  obligate  parasites.  Many, 
too,  have  been  noted  once  or  twice  only  in  certain  pathological 
conditions,  and  it  is  by  no  means  certain  that  they  were  more  than 
accidental  saprophytes. 

The  genera  of  molds  containing  species  of  known  pathogenicity 
are — 

Aspergillus. 

Penicillium. 

Fusarium. 

Sporotrickum. 

Microsporon  and  Trichophyton. 

Achorion. 

Oldium  or  Oospora. 

THE  GENUS  ASPERGILLUS 

The  Aspergilli  are  widely  distributed  in  nature.  They  are 
abundant  in  the  soil  and  on  decaying  materials  of  all  kinds. 
Their  spores  are  common  in  the  air,  and  cultures  may  readily  be 
secured  in  most  localities  by  simple  plate  exposure.  They  are  not, 
however,  present  in  such  numbers  as  the  genus  next  to  be  de- 
scribed, Penicillium.  Several  hundred  species  have  been  de- 
scribed, and  by  some  authors  the  genus  is  subdivided  into  two 
genera,  Aspergillus  and  Sterigmatocystis. 

Aspergillus  is  placed  by  the  botanists  among  the  Ascomycetes 
or  sac  fungi,  because  at  one  stage  in  the  life  history  sexual  repro- 
duction occurs,  resulting  in  the  formation  of  sacs  filled  with  spores. 
This  phase  of  the  life  history  has  been  worked  out  in  but  few  species; 


394 


VETERINARY   BACTERIOLOGY 


however,  it  is  probable  that  it  occurs  in  all  when  grown  under  t he- 
right  conditions. 

—The  mycelium  of  Aspergillus  is  colorless  and  hyaline,  'much- 
branched,  penetrating  for  a  short  distance  into  the  substratum  or 
medium,  and  usually  sending  up  aerial  hyphu>,  which  give  the 
colony  a  floccose  or  downy  appearance.  The  hyphse  are  septate, 
that  is,  cross  walls  are  formed  and  the  cells  are  divided  from  each 
other  by  walls.  Asexual  reproduction  takes  place  by  the  forma- 


Fig.  172. — Morphology  of  the  Aspergillus  glaucus:  a,  Mycelia  and  perithecia 
on  the  surface  of  the  medium,  with  a  single  conidiophore;  d,  a  very  young  peri- 
thecium;  e,  cross-section  through  a  perithecium,  somewhat  older;  /,  cross- 
section  through  a  mature  perithecium,  showing  the  asci  and  the  ascospores 
(as);  65l,  isolated  asci;  cc1,  ascospores  ripe  and  germinating  (c  bl  d  e  f  after 
deBary,  a  b  cl  after  Wehmer). 

tion  of  enlarged,  erect,  spore-bearing  hyphae  called  conidiophores. 
These  conidiophores  are  inflated  at  the  tip  and  become  covered  with 
papillae,  which  develop  into  short  stalks,  called  sterigmata  (singular, 
sterigma).  The  sterigmata  may  branch  once  or  many  times. 
giving  rise  to  bunches  of  secondary  sterigmata,  or  they  may  remain 
unbranched.  The  species  wit  h  1  > ranched  sterigmata  are  frequent  1  y 
grouped  together  into  a  genus  Sterigmatocystis.  From  the  tips  of 
these  sterigmata  spores  or  conidia  are  abjointed,  .-md  hanu  together 
to  form  long  chains.  The  spore  mass  at  the  tip  of  the  conidiophore 


MOLD  OR  HYPHOMYCETE  GROUP  395 

is  termed  a  head.  The  spores  are  usually  colored  green,  brown, 
yellow,  or  black,  or  in  a  few  species  they  are  colorless.  They  are 
spherical  or  oval  in  shape.  Their  surfaces  are  not  easily  wetted; 
they  are  easily  detached,  and  are  readily  carried  about  by  currents 
of  air.  This  explains  the  readiness  with  which  birds  and  some 
animals  become  infected  in  the  respiratory  tract  when  fed  on 
moldy  grain  or  fodder.  When  the  spores  come  under  favorable 
growth  conditions,  they  germinate  and  reproduce  the  mold. 

If  growth  conditions  are  right,  careful  observation  will  enable 
one  to  discover  the  sexual  stages  in  the  reproduction.  Two 
filaments,  somewhat  differentiated,  begin  to  twist  together  until 
they  form  a  typical  cork-screw.  The  cell  contents  fuse,  and  fer- 
tilization is  effected.  A  tangled  mass  of  threads  arises  about 
this  cell,  forming  a  compact  layer  or  covering  termed  the  peri- 
thecium.  The  inclosed  cell  grows  rapidly,  and  produces  a  con- 
siderable number  of  enlarged  cells,  each  of  which  eventually  is 
found  to  contain  spores,  usually  eight  in  number.  These  cells 
or  sacs  are  called  asci  (singular  ascus),  and  the  spores  are  termed 
ascospores.  These,  like  the  conidia,  when  brought  under  favorable 
growth  conditions,  reproduce  the  mold.  An  Aspergillus  may  con- 
tinue to  multiply  indefinitely  without  the  sexual  stage  developing; 
it  is  not  improbable  that  some  species  have  altogether  lost  the 
power  of  reproducing  other  than  by  means  of  the  conidia. 

Several  species  of  Aspergilli  have  been  described  as  pathogenic. 
Doubtless,  these  are  normally  saprophytes,  and  only  produce 
disease  under  exceptional  conditions. 

Aspergillus  fumigattrs 

Diseases  Produced. — Aspergillosis  of  birds;  pneumomycosis  in 
man  and  many  animals. 

The  occasional  presence  of  Aspergillus  in  lung  infections  has 
been  known  since  early  in  the  nineteenth  century.  Probably 
Mayer  and  Emmet,  in  1815,  were  the  first  to  note  its  presence  in 
the  lungs  of  a  bird,  in  this  instance  a  jay.  Since  that  time  the 
organism  has  been  reported  many  times.  In  most  cases  no  careful 
species  determination  was  made,  but  the  probabilities  are  greatly 
in  favor  of  Aspergillus  fumigatus  being  the  species  responsible. 
has  been  reported  from  the  stork,  raven,  flamingo, 


396 


VETERINARY    BACTERIOLOGY 


eider-duck,  parrot,  pigeon,  chicken,  hawk,  bullfinch,  plover, 
pheasant,  bustard,  duck,  goose,  ostrich,  swan,  and  turkey  among 
birds,  from  the  horse,  dog,  and  cow  among  animals,  and  from  man. 
It  has  been  reported  from  Europe  and  the  United  States. 

Morphology. — In  culture-medium  it  forms  greenish  or  bluish 
gray  or  later  brownish  masses.  The  conidiophores  are  abundant, 
but  short.  The  enlarged  tip  of  the  conidiophores  is  hemispherical, 
and  8  to  20  ^  in  diameter,  bluish-green,  and  later  brown.  The 


Fig.  173. — Aspergillusfumigatus:  1,  Optical  section  through  a  conidiophore; 
2,  conidiophore  and  conidia;  3,  conidia;  4,  a,  perithecium;  b,  an  isolated  ascus; 
c,  d,  ascospores,  front  and  lateral  view;  5,  swollen  hyphae,  bl,  and  conidiophores 
(4,  a-d,  after  Grijns,  remaining  after  Wehmer). 

perithecia  with  ascospores  have  been  observed  in  culture-media. 
In  the  lung  tissues  the  branching  mycelium  may  be  observed  on 
microscopic  examination,  and  the  sporophores  may  be  seen  pro- 
jecting into  the  air-sacs,  where  the  conidia  are  produced.  These 
spores  are  never  formed  except  in  the  presence  of  oxygen. 

Isolation  and  Culture. — The  Aspergillus  fumigatus  may  be 
readily  isolated  from  the  lesions  upon  almost  any  of  the  commonly 
used  artificial  media,  particularly  when  spores  are  produced.  For 


MOLD    OK    HYPHOMYCETE    GROUP  397 

the  best  development  the  medium  should  be  slight ly  acid.  It 
develops  readily  upon  potato  and  bread.  Colonies  become  visible 
in  a  day,  usually  as  tiny,  white,  cottony  growths,  which,  within 
n  few  days,  turn  green,  due  to  the  formation  of  the  spores  of  that 
color. 

Physiology. — The  Aspergillus  fumigatus  is  an  aerobe.  The 
optimum  growth  temperature  is  from  35°  to  40°.  According  to 
Mohler  and  Buckley,  growth  occurs,  but  spores  do  not  form 
below  20°.  The  spores  are  resistant  to  high  temperatures.  They 
have  been  found  to  survive  an  exposure  of  seven  hours  at  65°. 


Fig.  174. — Aspergillus  fumigatus  from  a  culture  on  agar  (Frankel  and  Pfeiffer). 

Twelve  hours'  contact  with  a  5  per  cent,  solution  of  phenol  is  not 
sufficient  certainly  to  destroy  them.  They  can  withstand  desicca- 
tion indefinitely. 

Pathogenesis. — Experimental  Evidence. — The  organism  pro- 
duces death  within  a  few  hours  or  days  when  injected  intravenously 
or  intrathoracically  into  the  chicken.  The  pigeon  is  particularly 
susceptible  to  injections.  Rabbits  and  guinea-pigs  likewise  suc- 
cumb, usually  from  a  generalized  infection.  There  is  sufficient 
experimental  evidence  to  justify  the  conclusion  that  Aspergillus 
fumigatus  may  produce  a  primary  and  fatal  infection  in  man}' 
animals. 


398  VETERINARY  BACTERIOLOGY 

Character  of  Disease  and  Lesions  Produced. — By  far  the  greatest 
number  of  cases  of  aspergillosis  have  been  reported  from  birds. 
The  lesions  are  generally  located  in  the  lungs,  air-sacs,  and  hollow 
bones,  where  the  spores  may  readily  lodge.  In  man  and  animals, 
particularly  the  horse,  the  usual  picture  is  an  infection  of  the  lungs 
and  air-passages,  but  occasionally  of  the  mucous  membranes  of 
other  parts  of  the  body.  Metastatic  infection  of  other  organs  is 
not  infrequent.  The  organism  causes  the  development  of  nodules 
not  unlike  those  of  tuberculosis.  In  the  lungs  the  tubes  are  fre- 
quently occluded  by  the  green  fructifications  of  the  fungus.  There 
is  more  or  less  necrosis  of  tissue  immediately  surrounding  the  or- 
ganisms. 

Immunity. — Several  investigators  claim  to  have  produced  toxic 
substances,  if  not  true  toxins,  by  the  growth  of  the  organisms  in 
artificial  media.  These  claims  have  not  been  sufficiently  sub- 
stantiated, although  there  is  considerable  a  priori  evidence, 
from  the  character  of  the  lesions  and  symptoms,  that  powerful 
toxic  substances  of  some  kind  are  produced.  No  method  of  suc- 
cessful immunization  has  been  developed. 

Bacteriological  Diagnosis. — A  diagnosis  may  usually  be  made 
by  the  character  of  the  lesions  and  the  appearance  of  the  green 
spores.  A  microscopic  examination  of  the  scrapings  of  the  in- 
fected mucous  membranes  should  reveal  the  spores  and  character- 
istic conidiophores  without  difficulty. 

Transmission. — The  organism  doubtless  grows  on  decaying 
organic  matter  outside  the  body.  The  feeding  of  moldy  grain  or 
fodder  may  give  ample  opportunity  for  infection  by  inhalation. 
The  preponderance  of  pulmonary  primary  infections  shows  that 
inhalation  probably  is  the  common  method  of  infection. 

Aspergillus  flavus 

This  organism  has  been  described  by  various  investigators, 
who  believed  it  to  be,  in  part  at  least,  responsible  for  blind  staggers 
or  meningoencephalitis  in  horses.  It  occurs  in  great  quantities 
on  moldy  corn  and  other  grains.  Although  the  complete  data 
have  not  been  presented,  the  work  of  Haslam  indicates  that  it  is 
of  some  pathogenic  significance. 

Morphology. — The  sterile   hyphae  are   cobwebby  and   white. 


MOLD  OR  HYPHOMYCETE  GROUP  399 

The  conidiophores  are  erect.     Conidia  are  5  to  7  p  in  diameter, 


Fig.  175. — Aspergittus  flavus:  1,  2,  3,  4,  Various  stages  in  the  development 
of  conidiophores;  5,  section  and  surface  of  a  hypha,  showing  the  numerous 
colorless  granules  with  which  it  is  covered;  6,  natural  size  of  the  fungus;  7, 
conidia  (Wehmer). 

globose.     Spore  masses  are  yellow  and  yellowish  green.     Sclerotia 
are  small  and  dark. 

Aspergillus  niger 

This  organism  occurs  under  conditions  similar  to  the  preceding, 
and  is  believed  also  to  be  pathogenic  to  horses  and  other  animals 
that  consume  grain  infected  with  it. 

Morphology. — The  mycelium  is  at  first  white,  then  darker, 
abundant,  and  penetrates  the  medium  to  a  considerable  distance. 
The  conidiophores  are  long  and  the  spores  borne  up  at  some  dis- 
tance from  the  surface  of  the  substratum.  The  sterigmata  are 
branched.  The  conidia  are  3.5  to  4.5  ^  in  diameter,  roughened. 
The  spore  masses  ultimately  become  black,  and  may  be  readily 
differentiated  in  this  manner  from  the  two  preceding.  This 


400 


VETERINARY   BACTERIOLOGY 


Fig.  176. — AspergiUus  niger:  1,  2,  3,  4,  Stages  in  the  development  of  the  con- 
idiophores;  5,  conidia;  6,  detail,  showing  the  branched  sterigmata;  7,  S. 
sclerotia;  9,  natural  size  of  the  fungus  (Wehmer). 

organism  has  also  been  found  in  the  ear  and  in  lesions  in  the 
lungs. 

Other   Species  of  Aspergilli 

Several  other  species  of  Aspergilli  have  been  reported  as 
pathogenic.  Among  them  are  AspergiUus  nigrescens,  A.  subfuscus, 
and  A.  glaucus.  These  produce  nodular  mycotic  foci  in  the  in- 
ternal organs  of  laboratory  animals  into  which  they  have  been 
injected.  They  do  not  commonly  produce  infection  under  natural 
conditions. 

THE  GENUS  PENICILLIUM 

Penicillium  is  closely  related  to  AspergiUus,  the  principal 
difference  being  the  manner  in  which  the  asexual  >p<»n •>  or  conidia 
are  borne.  The  conidiophores  are  erect,  and  much  branched  at  the 
tip,  the  branches  arising  in  whorl-  ;in«l  arc  not  enlarged  at  the  apex. 
From  the  end  of  each  ultimate  branch  a  chain  of  spores  is  abjoint  cd. 


MOLD    OR   HYPHOMYCETE    GROUP 


401 


giving  to  the  organism  under  the  microscope  the  appearance  of 
being  covered  with  little  brooms.  The  sexual  stage  is  essentially 
similar  to  that  of  Aspergillus.  Penicillia  are  even  more  common 
than  the  Aspergilli.  They  occur  as  blue  or  green  molds  upon  fruit, 
and  upon  a  great  variety  of  decaying  materials.  Of  the  hundreds 
of  species  of  Penicillium  that  have  been  described,  the  majority 


Fig.  177. — Penicillium  glaucum:  a,  a1,  Tips  of  conidiophores  showing  the 
characteristic  method  of  branching  and  the  chains  of  spores;  b,  perithecium ; 
c,  asci;  d,  ascospores  (Brefeld). 

are  green  or  bluish-green  in  color,  but  white,  gray,  yellow,  orange, 
and  brown  forms  are  known. 

None  of  the  species  of  Penicillium  are  known  to  be  harmful, 
but  their  constant  presence  in  moldy  silage  and  grain  which  has 
poisoned  animals  makes  it  necessary  to  consider  them  in  any 
discussion  of  forage  poisoning. 

THE  GENUS  FUSARIUM 

The  members  of  this  genus  are  nearly  all  saprophytes  or  plant 
parasites.     Fusarium   is   included   among  the   Fungi   imperfecti, 
as  a  sexual  or  perfect  stage  is  unknown  in  the  life  history  of  most 
26 


402 


VETERINARY    BACTERIOLOGY 


of  the  species.  Webber  has  found  an  ascus  stage  in  one  species, 
and  concludes  that  the  genus  Necomospora  of  the  Ascomycetes  is 
the  perfect  form;  in  other  species  it  is  the  genus  Gibberetta. 

Fusarium  is  characterized  by  its  loose,  spreading,  cottony 
mycelium  with  numerous  cross  walls,  i.  e.,  septate.  The  conidio- 
phores  are  not  markedly  different  from  the  sterile  hyphae,  and  are 
usually  branched.  The  conidia  are  borne  at  the  tips  of  these 
branches.  They  are  long,  slender,  sickle  or  crescent-shaped 


Fig.  178. — Fusarium  corallinum,  conidiophores  and  conidia  (Saccardo). 

usually,  and  divided  into  several  or  many  cells  by  cross  walls  or 
septa. 

Several  species  of  Fusarium  are  found  commonly  on  grains  and 
moldy  corn.  This  fungus  has  been  believed  by  some  investigators 
to  be  of  significance  in  forage  poisoning.  It  is  one  of  the  several 
forms  which  must  be  considered  in  a  determination  of  the  poisonous 
properties  of  forage. 

One  species,  the  Fusarium  equinum,  is  believed  to  produce 
dermatitis  in  the  horse. 

Fusarium  equinum 

Disease  Produced. — Itch  disease,  associated  with  sarcoptio 
dermatitis. 

Norgaard,  in  1901,  noted  the  presence  of  a  Fusarium  in  adenim- 
titis  of  horses  in  the  State  of  Oregon,  and  proposed  the  name 
Fusarium  equinum  for  the  fungus.  Melvin  and  Mohler  later 
studied  the  disease  in  greater  detail.  The  disease  has  been  re- 


MOLD   OR   HYPHOMYCETE   GROUP 


403 


ported  only  from  this  one  locality,  but  in  this  instance  affected 
several  thousand  horses  on  the  Umatilla  Indian  reservation. 

Morphology. — The  mycelium  upon  culture-media  is  septate  and 
branched.  Three  forms  of  spores  are  produced.  The  microconidia 
are  small  and  oval,  one-  or  two-celled.  The  macroconidia  are 
large,  sickle-shaped,  three  to  five  septate,  and  pointed  at  the 
ends.  They  are  25  to  55  U  long  by  2.5  to  4.5  u  wide.  Chlamydo- 
spores  are  formed  in  the  mycelial  threads,  by  a  cell  rounding  up  to 


Fig.  179. — Fusarium  equinum,  mycelium  and  conidia  (Melvin  and  Mohler, 
Bureau  of  Animal  Industry). 

a  diameter  of  8  to  15  fi  and  becoming  densely  granular.  The 
spores  may  be  recognized  in  the  hair-follicles  of  the  diseased  ani- 
mals. 

Isolation  and  Culture. — Xo  difficulty  was  experienced  in  secur- 
ing a  growth  of  the  organism  on  artificial  media.  The  more 
favorable  media  are  potato  and  bread,  but  good  growth  will  take 
place  on  glucose  or  plain  agar.  The  growth  is  white  and  cottony, 
and  the  spores  are  produced  in  abundance. 

Pathogenesis. — Inoculation  experiments  were  unsuccessful,  so 


404  VETERINARY   BACTERIOLOGY 

that  the  evidence  of  pathogenesis  rests  entirely  upon  the  constant 
occurrence  of  the  organism  in  the  disease  in  question.  Itch- 
mites  (Sarcoptes  equi)  were  found,  but  the  investigators  believe 
their  numbers  insufficient  to  account  for  the  disease.  It  is  entirely 
possible  that  the  organism  is  a  secondary  invader,  or  produces  the 
disease  in  a  kind  of  symbiotic  relationship  with  the  Sarcoptes. 
The  fungus  seems  to  enter  the  hair-follicles,  penetrates  between 
the  epidermal  cells,  and  involves  the  surrounding  skin,  causing 
an  intense  itching.  The  body  becomes  covered  with  a  crust  or 
scurf,  at  first  gray  and  afterward  darker.  The  presence  of  the 
organism  in  the  hair-follicles  causes  the  hairs  to  fall  out,  resulting 
in  an  almost  complete  alopecia. 

THE  GENUS  SPOROTRICHUM 

Authorities  differ  greatly  in  the  delimitation  of  this  genus. 
According  to  botanists,  the  genera  Microsporon  and  Trichophyton 
are  synonyms  of  Sporotrichum.  Pathologists  and  bacteriologists 
in  general,  however,  make  a  distinction  between  them.  The 
classification  of  the  latter  will  be  adopted  here  and  the  term  Sporo- 
trichum used  in  the  narrow  sense. 

Sporotrichum  is  distinguished  by  the  production  of  definite 
hyphae,  which  are  usually  creeping  and  irregularly  branched. 
Definite  conidiophores  are  not  developed,  or  consist  only  of  small 
side  branches.  The  conidia  are  borne  either  on  the  sides  or  ends 
of  the  hyphae,  singly  or  in  clusters.  They  are  usually  very  numer- 
ous, ovoid  or  spherical  in  shape,  and  hyaline  or  rarely  lightly 
colored.  The  molds  belonging  to  this  group  are  in  need  of  careful 
study  and  revision,  as  there  is  great  uncertainty  concerning  many 
of  the  species. 

One,  or  possibly  two,  species  of  Sporotrichum  have  been  shown 
recently  to  be  of  considerable  pathogenic  significance. 

Sporotrichum  beurmanni 

Synonym. — Fossil >ly  Sporotrichum  schenkii. 

Disease  Produced.  Sporotrichosis  in  man  and  animals,  one 
typi!  of  epizootic  lymphangitis  in  horses. 

Schenk,  in  1898,  and  Hoktoen  and  Perkins,  in  1900,  described 
a  species  of  Sporotrichum  causing  multiple  abscesses  in  man. 


MOLD   OR  HYPHOMYCETB   GROUP 


405 


de  Beurnuinn,  in  1903,  described  similar  forms  from  France.  The 
disease  has  been  reported  in  man  and  rats  from  Brazil,  from  man 
in  California,  Argentina,  Germany,  and  in  the  horse  in  the  United 
States  and  Madagascar.  This  disease  is  probably  quite  wide-spread, 
but  has  not  been  recognized,  or  has  been  confused  with  others, 
until  recently.  It  is  to  be  differentiated  sharply  from  the  true 
epizootic  lymphangitis  of  the  horse.  An  excellent  discussion  of 
the  organism  in  its  relation  to  disease  in  the  horse  was  contributed 
by  Page,  Frothingham,  and  Paige  in  1910. 


Fig.  180. — Sporotrichum  beurmanni,  from  culture  showing  the  mycelium  and 
spores  (Page,  Frothingham,  and  Paige,  in  "  Journal  of  Medical  Research"). 

Morphology. — The  examination  of  the  material  from  culture  is 
most  easily  made  in  a  hanging  drop.  The  hyphse  are  slender  and 
septate.  Spores  or  conidia  are  borne  at  the  tips  of  side  branches; 
usually  a  number  are  formed  successively  and  are  found  then 
in  clusters.  The  conidia  are  small,  oval  or  spherical.  They  fre- 
quently bud  to  some  extent,  and  resemble  somewhat  the  cells  of 
Blastomyces.  The  hyphse  stain  easily  with  the  common  anilin 
dyes  and  are  gram-positive.  In  using  the  latter  stain  the  alcohol 
must  not  remain  too  long  in  contact,  otherwise  the  stain  will  be 
removed.  Whether  or  not  the  organism  ever  develops  a  perfect 
or  sexual  stage  is  not  known,  but  it  does  not  seem  probable. 


406 


VETERINARY   BACTERIOLOGY 


Isolation  and  Culture. — The  organism  may  readily  be  isolated 
from  pus  from  the  lesions.  Potato  is  the  most  favorable  medium. 
Original  isolations  show  at  the  end  of  a  week,  transplants  at  the 
end  of  two  or  three  days,  as  white,  filamentous  colonies.  These 
enlarge,  become  darker  at  the  center,  and  finally  turn  dark 
brown  or  black,  frequently  surrounded  by  a  rim  of  white.  The 

colony  becomes  wrinkled.  Upon  gela- 
tin the  growth  remains  white.  Lique- 
faction begins  in  from  three  to  ten 
days  or  even  later.  The  addition  of 
dextrose  causes  the  center  of  the  col- 
onies to  darken.  In  agar,  and  partic- 
ularly in  neutralized  glycerin  agar, 
growth  is  good,  and  the  colonies 
remain  white.  Blood-serum  is  not 
liquefied.  In  litmus  milk  growth 
occurs  with  little  change  in  the 
medium,  or  coagulation  without  acid 
production  may  take  place  after  the 

jjjhjff'  lapse    of    several    weeks.      In    liquid 

J  media  growth  occurs  in  the  form  of 

4  more  or  less  separated  colonies,  usually 

accompanied  by  a  surface  pellicle. 

Physiology. — The  organism  is  an 
obligate  ae'robe.  Growth  occurs  best 
at  25°  to  28°,  but  is  not  prevented 

Fig.  ISl.-Sporotrichum  at  37°'  The  spores  resist  desicra- 
beurmauni,  cultures  and  col-  tion  for  considerable  periods.  Acid 
onies  on  potato  (Page,  Froth-  js  produced  from  dextrose,  but  not 

•tzk, " d  M£&  R:  f—  I-*-. mait-.  ~* h— - — 

search").  n^e>    dulcit,    adonit,    inulin,    or    raf- 

finose.      Gas    is    not    produced    from 

any  sugar.  No  indol  is  formed.  Gelatin  is  slowly  liquefied. 
The  organism  is  destroyed  at  a  temperature  of  60°  for  five 
minutes. 

Pathogenesis. — Experimental  Evidence. — There  is  an  abundance 
of  evidence  that  Sporotrichwn  beurmanni  is  pathogenic  for  man  and 
animals.  Accidental  laboratory  infections  have  taken  place  in 


MOLD   OR   HYPHOMYCETE   GROUP  407 

man.  Inoculation  of  pure  cultures  into  mice  and  white  rats 
liives  rise  to  abscesses  at  the  point  of  inoculation,  and  the  infec- 
tion gradually  extends.  Guinea-pigs  and  rabbits  are  infected 
with  greater  difficulty.  An  infection  somewhat  resembling  farcy 
develops  upon  inoculation  into  the  horse. 

Character  of  Disease  and  Lesions  Produced. — The  infection  is 
usually  benign  in  character  in  the  horse.  There  is  no  fever  reac- 
tion during  the  course  of  the  disease.  Nodules  develop  which  are 
generally  spherical  and  sharply  delineated.  These  nodules  scarcely 
rupture,  but  pus  accumulates  at  the  center,  the  skin  above  is 
thinned  and  softened,  serum  exudes  from  the  surface,  the  hair  is 
loosened,  and  a  crust  holding  the  hairs  together  is  formed.  The 
ulcers  are  crateriform,  and  usually  contain  a  little  creamy  pus. 
Healing  is  accomplished  by  granula- 
tion. Sections  of  nodules  from  labor- 
atory  animals  reveal  the  organism  in 
the  tissues. 

Immunity. — No  toxins  have  been 
demonstrated  for  this  organism. 
Widal  has  shown  the  presence  of 
agglutinins  for  the  spores  in  the 
blood  of  infected  individuals.  No  Fig.  182.  —  Sp&rotrichum 
method  of  immunization,  based  upon  teurmanm,  in  a  section  of  a 
the  organism  or  its  products,  has  mesenteric  abscess  of  a  rat 

(adapted  from  Fiehtz). 
been  developed. 

Bacteriological  Diagnosis. — This  may  be  accomplished  by 
animal  inoculation,  thus  securing  the  organism  in  pure  culture. 
Widal  claims  that  the  agglutination  of  spores  will  take  place  in 
dilutions  as  high  as  1:800,  but  the  same  reaction  takes  place  in 
lower  dilutions  with  the  blood-serum  of  individuals  having  ac- 
tinomycosis.  Bloch  found  that  a  bouillon  filtrate  from  an  old 
culture  would  in  man  give  the  von  Pirquet  cutaneous  reaction,  as 
was  described  in  the  chapter  on  Bacillus  tuberculosis. 

Transmission. — The  disease  is  transmitted  by  intimate  contact 
usually.  It  has  been  found  to  be  transferred  from  animals  to  man. 
Probably  the  organism  usually  gains  entrance  through  abrasions  of 
the  skin. 


408  VETERINARY    BACTERIOLOGY 

THE  GENERA  TRICHOPHYTON  AND  MICROSPORON,  ACHORION, 

AND   OlDIUM 

The  organisms  belonging  to  the  two  genera,  Trichophyton  and 
Microsporon,  are  frequently  included  together  under  the  single 
genus  Sporotrichum.  The  two  names,  Trichophyton  and  Micro- 
sporon, are  also  used  very  loosely  and  interchangeably.  A  care- 
ful study  of  the  relationships  of  the  various  forms  is  needed. 

No  very  clear  differentiation  of  this  group  from  the  preceding 
can  be  given. 

Organisms  belonging  to  these  genera  are  the  cause  of  many 
skin  and  hair  infections  in  man  and  animals.  Pityriasis  versicolor, 
or  tinea  versicolor,  in  man  is  caused  by  Microsporon  furfur 
(Sporotrichum  furfur) ;  herpes  tonsurans,  or  ringworm  in  man  and 
lower  animals,  by  Trichophyton  tonsurans.  Other  species  have  been 
described  from  most  of  the  domestic  animals  and  birds,  but  they 
have  not  in  most  cases  been  sufficiently  denned  and  differentiated. 

Trichophyton  tonsurans 

Synonyms. — The  synonymy  of  the  various  forms  included 
under  this  name  is  almost  inextricable.  Probably  more  than  one 
good  species  is  represented.  The  commoner  synonyms  are 
Trichophyton  microsporon;  Tr.  megalosporon ;  Microsporon  adouini; 
M.  equinum;  M.  carinum;  Sporotrichum  tonsurans;  S.  adouini,  and 
many  others. 

Disease  Produced. — Herpes  tonsurans,  or  ringworm,  in  man, 
goat,  sheep,  pig,  horse,  cat,  and  dog. 

The  organism  was  originally  described  by  Gruby  in  1842.  In 
its  various  forms  it  is  world  wide  in  distribution. 

Morphology. — The  hyphae  are  relatively  slender,  septate;  the 
spores  are  numerous  in  the  hairs  and  scales.  They  vary  in  the 
different  types  described  from  2  to  8  u  in  diameter.  Chlamydo- 
spores  are  produced  in  artificial  media. 

Isolation  and  Culture. — Pure  cultures  are  secured  with  diffi- 
culty, as  the  skin  and  hair  are  generally  filled  with  bacteria  which 
overgrow  the  mold.  Krai  has  suggested  pulverizing  hairs  with 
fine  sand  or  silicon  powder  and  pouring  gelatin  plates  of  various 
dilutions.  Kitt  has  taken  advantage  of  the  resistance  of  the 
organism  to  destruction  by  alkalies  by  washing  the  hairs  and 


MOLD  OR  HYPHOMYCETE  GROUP  409 

scales  from  an  infected  area  with  a  solution  of  KOH,  which  removes 
most  of  the  bacteria  without  materially  injuring  the  mold  spores. 
Potato  is  a  favorable  medium  for  growth.  A  rather  velvety, 
wrinkled,  relatively  heavy,  membrane  forms  which  may  be  white 
or  colored.  Gelatin  is  slowly  liquefied. 


Fig.  183. — Trichophyton  tonsurans  from  agar  plate  culture  (Giinther). 

Pathogenesis. — The  disease  in  all  animals  is  characterized  by 
the  formation  of  scales,  the  falling  out  of  hair,  with  or  without 
suppuration.  It  is  well  demonstrated  that  it  can  spread  from 
one  species  of  animal  to  another  by  contact. 

Achorion  schoenleinii 

Synonyms. — O'idium  porriginis;  Oospora  porriginis,  and  many 
described  species  of  Achorion.  Opinions  differ  as  to  whether 
one  polymorphic  species  or  many  species  are  represented. 

Diseases  Produced. — Favus,  tinea  favosa,  in  man,  animals, 
and  birds. 

The  organisms  grouped  under  this  name  have  been  separated 
into  a  large  number  of  species  by  some  investigators.  Doubtless 
more  than  one  species  should  be  recognized,  but  they  have  not 
been  sufficiently  differentiated. 

Morphology. — The  organism  is  made  up  of  more  or  less  branched 
hyphse,  3  to  5  f*  in  diameter.  Spores  are  formed  by  the  breaking 
up  of  the  filaments  into  oval  or  somewhat  cubical  spores,  3  to  8 


410  VETERINARY   BACTERIOLOGY 

by  3  to  4  it  in  diameter.     The  mycelium  may  be  quite  luxuriant 
in  the  skin. 

Pathogenesis. — The  disease  is  characterized  by  the  formation 


Fig.  184. — Achorion   schoenleinii,  section  showing  the  hyphae  (Friinkel  and 

Pfeiffer). 

of  scales  upon  the  skin,  often  accompanied  by  a  disagreeable  odor. 
It  may  be  communicated  from  one  species  of  animals  to  another. 

Oi'dium  albicans 

Synonym. — Monilia  Candida. 

Disease  Produced. — Thrush  in  infants;  sometimes  in  the  young 
of  animals. 

Berg,  in  1840,  described  this  organism  as  the  cause  of  thrush. 
It  has  since  that  time  been  repeatedly  isolated  and  cultivated. 
It  is  known  from  most  civilized  countries. 

Morphology. — The  mycelium  of  this  organism  is  poorly  de- 
veloped; frequently  the  whole  growth  consists  of  budding  yeast- 
like  cells.  These  may  be  spherical,  elliptical,  oval  or  cylindrical, 
the  shorter  cells  about  4  p  in  diameter  by  5  to  6  ^  in  lengt  h.  The 
hyphal  threads  are  much  longer.  There  can  be  little  differentia- 
tion in  many  cases  between  the  conidia  and  the  cells  of  the  hyplue. 
but,  on  artificial  media,  the  conidia  are  frequently  definitely  ad- 


MOLD  OR  HYPHOMYCETE  GROUP  411 

jointed  from  the  tip  of  the  conidiophore.  Chlamydospores  may 
form  in  the  hyphae. 

Isolation  and  Culture. — The  organism  may  be  isolated  without 
difficulty  from  the  lesions  of  the  disease.  A  distinctly  acid  medium 
should  be  used.  On  most  nutrient  media  it  develops  superficial, 
spherical,  white,  waxy  to  granular  colonies.  Gelatin  is  not 
liquefied,  nor  is  blood-serum. 

Pathogenesis. — The  organism,  when  injected  intravenously 
into  rabbits,  produces  a  fatal  infection,  not  unlike  a  generalized 
infection  with  a  Blastomyces.  Typical  thrush  has  been  pro- 
duced in  the  mouth  of  young  animals  and  birds  by  inoculation. 
The  disease  generally  occurs  in  the  mouth  of  sucklings,  usually  as 
a  benign  infection.  It  is  characterized  by  the  formation  of  white 
patches  on  the  mucous  membrane,  varying  in  size  from  points  to 
considerable  areas.  The  infection  may  extend  to  the  pharyngeal 
or  laryngeal  mucosse;  rarely  metastatic  infection  of  internal 
organs  may  occur. 


SECTION  V 
PATHOGENIC  PROTOZOA 

CHAPTER  XXXIX 

STRUCTURE,  RELATIONSHIPS,   AND   CLASSIFICATION    OF    THE 

PROTOZOA 

A  PROTOZOA x  may  be  defined  as  a  unicellular  organism  be- 
longing to  the  animal  kingdom.  Protozoa  exist  throughout  their 
life-history  as  single-celled  individuals,  or  as  colonies  of  single 
cells;  that  is,  the  cells  are  not  united  to  form  tissues  or  organs, 
and  never  constitute  a  portion  only  of  a  multicellular  form. 

The  protozoa  show  some  forms  which  intergrade  with  the  bac- 
teria. The  group  containing  the  spiral  forms,  such  as  the  Spiro- 
chaeta,  is  at  present  a  questionable  one,  some  investigators  be- 
lieving that  it  has  more  bacterial  than  protozoan  characteristics, 
others  taking  quite  the  opposite  view.  It  is  difficult,  on  the  other 
hand,  to  draw  a  sharp  line  of  demarcation  between  the  protozoa 
and  the  multicellular  animals  or  metazoa.  Just  when  a  group  of 
cells  ceases  to  be  a  mere  group  of  independent  units,  and  becomes 
a  tissue  which  forms  the  whole  or  part  of  a  multicellular  form,  it  is 
difficult  to  determine. 

Although  the  protozoa  are  regarded  as  the  simplest  and  most 
primitive  of  living  things,  nevertheless  many  are  complex  in  struc- 
ture and  have  the  cell  divided  into  specialized  parts,  sometimes 
termed  organella,  somewhat  similar  in  function  to  the  organs  of  the 
higher  forms.  They  frequently  undergo  many  changes  in  form 
during  their  life.  Their  life-history  is,  thcn-forr,  relatively  com- 
plex as  compared  with  that  of  the  bactrrm. 

Structure  of  the  Protozoa. — The  body  substance  of  a  protozoan 
may  be  divided  into  the  ectoplasm, or  outer  layer,  which  comes  into 


CLASSIFICATION    OF   THE    PROTOZOA  413 

contact  with  the  material  environment,  and  the  endoplasm,  with- 
in. Within  the  endoplasm  there  is  generally  a  nucleus  (in  some 
forms  two,  a  large  and  a  small) ,  and  frequently  inclusions  of  other 
sorts. 

The  ectoplasm  in  some  cases  cannot  be  differentiated  sharply 
from  the  endoplasm.  These  are  the  exception,  however,  and  not 
the  rule.  The  ectoplasm  may  become  variously  modified,  and,  by 
secretion,  form  shells  or  a  heavy  membrane  for  protection.  Many 
of  the  pathogenic  protozoa  become  encysted  by  the  formation  of  a 
heavy,  chitinous  wall. 

The  endoplasm  is  made  up  of  a  very  delicate,  foam-like  or 
alveolar  structure,  the  density  of  which  varies  greatly.  It  may 
inclose  amorphous  granules  or  crystals  of  different  kinds,  green 
granules  or  chromatophores,  vacuoles,  etc. 

None  of  the  protozoa  are  certainty  known  to  be  entirely  des- 
titute of  nuclear  material.  The  nucleus  may  be  devoid  of  a 
nuclear  membrane  or  distributed;  generally  a  membrane  is  pres- 
ent. A  single  homogeneous  nucleus  is  present  in  most  forms, 
with  the  exception  of  the  infusoria,  which  have,  in  general,  a  large 
or  macronudeus  and  a  small  or  micronucleiLS.  At  some  stages  in 
the  life-history  many  nuclei  may  be  present  in  a  single  cell.  This 
is  particularly  true  just  before  or  during  spore  formation. 

The  protozoa  with  few  exceptions  are  motile,  at  least  during 
certain  stages  in  the  life  history.  The  exceptions  are  among 
certain  of  the  parasitic  Sporozoa.  Special  organs  of  locomotion 
are  frequently  found.  Pseudopodia  are  changeable  processes  of 
the  protoplasm  which  are  thrown  out  by  the  cell,  the  cell  contents 
frequently  flowing  forward  into  the  pseudopodia.  Many  or  few 
may  be  present  at  one  time.  They  may  be  short  and  blunt  or 
long  and  slender.  Usually  the  endoplasm  as  well  as  ectoplasm 
takes  part  in  their  formation.  The  protozoan  flagella  are  derived 
from  the  ectoplasm;  are  usually  unchangeable  in  shape,  long, 
thin,  and  pointed;  in  general,  longer  than  the  cell  itself. 
One,  two,  or  many  may  be  present.  They  propel  the  cell  by  a 
series  of  undulations  or  spiral  or  rotary  motions.  Cilia,  when 
present,  are  found  in  considerable  numbers.  They  are  shorter, 
generally  blunt,  and,  by  striking  the  water  in  unison,  resemble 
oars  in  their  motion. 


414  VETERINARY   BACTERIOLOGY 

The  life  histories  of  the  various  protozoa  show  such  com- 
plexities that  they  may  better  be  treated  under  the  separate  sub- 
divisions. 

Classification  of  the  Protozoa. — Authorities  are  not  in  entire 
accord  with  reference  to  the  principal  subdivisions  of  the  protozoa. 
The  classes  here  used  are  those  given  by  Calkins  in  "  The  Pro- 
tozoa." The  definitions  of  the  groups  here  given  will  not  hold  in 
all  cases  for  non-parasitic  forms. 

CLASSES  OF  PROTOZOA 

A.  Motile  in  adult  life  by  means  of  pseudopodia.      Reproduc- 
tion by  simple  division  and  by  spore  formation.     I.  Sarcodina  or 
Rhizopoda. 

B.  Not  possessing  pseudopodia  in  the  adult  form. 

(1)  Adult  motile  by  means  of— 

a.  Flagella.     With  one  nucleus.     II.  Mastigophora. 

b.  Cilia.     Two  types  of  nuclei.     III.  Infusoria  or  Cilio- 
phora. 

(2)  Adult     forms     non-motile.     Reproduction    by    spores. 
IV.  Sporozoa. 

Pathogenic  organisms  belonging  to  each  of  the.four  classes  are 
known.  These  will  be  treated  under  separate  chapters. 


CHAPTER   XL 

PATHOGENIC  PROTOZOA  OF  THE  CLASS   SARCODINA 
(RHIZOPODA) 

THE  Sarcodinse  (Rhizopodse)  are  protozoa  having  during  adult 
life  movable  or  changeable  processes  of  protoplasm  called  pseudo- 
podia.  Reproduction  is  accomplished  through  simple  fission  and 
by  spore  formation. 

Among  the  hundreds  of  genera  and  thousands  of  species  of 
organisms  belonging  to  this  group  that  have  been  described  there 


V 

Fig.  I'v"). — Ameba  in  culture  (Schardinger). 

is  one  (possibly  two  or  three)  which  has  been  found  to  be  patho- 
genic.    They  are  certainly  known  to  be  pathogenic  in  man  only, 

41  r, 


410  VETERINARY  BACTERIOLOGY 

but  the  frequent  occurrence  of  the  organisms  of  this  genus  in  the 
intestines  of  the  lower  animals,  and  the  possibility  of  confusing 
the  adult  stage  with  certain  developmental  stages  of  the  sporozoa, 
renders  a  discussion  of  these  forms  advisable  in  a  veterinary  text. 
The  possibility  of  any  of  the  forms  being  pathogenic  for  lower 
animals  has  not  been  sufficiently  investigated. 

THE  GENUS  ENTAMOEBA 

The  normal  inhabitant  of  the  intestinal  tract  of  man  was 
first  known  as  Amoeba  coli.  Later,  Schaudinn  renamed  it  Enta- 
mceba  coli,  and  gave  the  name  Entamceba  histolytica  to  the  patho- 
genic type  associated  with  the  amebic  dysentery.  The  genus 
Entamoeba  was  differentiated  from  Amoeba  by  the  absence  of  a 
contractile  vacuole  and  the  formation  of  multinucleated  cysts. 

Authorities  differ  greatly  in  their  estimates  of  the  number  of 
species  of  amebae  present  in  the  intestines  of  man.  The  best 
classification,  and  the  one  most  commonly  used  now,  is  that  of 
Schaudinn.  He  recognizes  two  species  at  least — one  a  normal 
non-pathogenic  form,  Entamoeba  coli,  and  one  pathogenic  for  man, 
Entamoeba  histolytica.  More  recently  Hartmann  and  others  have 
described  a  third  species,  E.  tetragena,  and  Koidsumi  a  fourth, 
E.  nipponica,  both  from  cases  of  dysentery.  The  possibility  of 
any  of  the  dysenteries  of  the  lower  animals  being  caused  by  amebse 
has  not  been  sufficiently  studied.  It  was  at  one  time  believed  that 
certain  diseases  of  turkeys  and  other  fowls,  particularly  entero- 
hepatitis,  were  caused  by  an  ameba  termed  Amoeba  meleagridis,  but 
this  has  been  shown  to  be  but  a  developmental  stage  in  the  life 
history  of  a  Coccidium. 

Examination  of  Living  Amebce. — The  amebae  may  be  examined 
in  the  stools  in  a  living  condition,  by  placing  a  portion  of  the  liquid, 
or  a  bit  of  the  solider  material  moistened  with  physiological 
salt  solution  on  a  slide,  and  pressing  down  a  cover-glass  not  too 
firmly.  Craig  advises  the  use  of  a  very  weak  solution  of  neutral 
red  to  stain  the  living  organisms  when  they  are  not  present  in 
too  great  numbers.  For  the  specific  determination  of  the  amebae 
present  an  examination  of  this  kind  is  frequently  all  that  is  neces- 
sary. The  slide  must  be  maintained  at  about  blood  heat  in  order 
to  detect  motility. 


PATHOGENIC  PROTOZOA  OF  THE  CLASS  SARCODINA    417 

Staining  Methods. — The  organisms  stain  rather  readily  with  the 
usual  laboratory  stains,  but  these  are  of  little  value  in  the  differ- 
entiation of  the  parts  of  the  cell  and  in  separating  species.  Wright's 
stain  in  favorable  specimens,  if  carefully  used,  gives  the  best 
differentiation  of  the  parts  of  the  cell.  The  organism  in  tissues 
is  best  stained  by  Heidenhain's  iron-hematoxylon  or  Borrel's 
stain.  The  smears  should  be  fixed  from  fifteen  to  twenty  minutes 
in  alcohol  and  ether,  equal  parts,  for  all  stains  but  Wright's.  For 
the  latter  no  fixation  is  required. 

Methods  of  Isolation  and  Cultivation. — Amebae  commonly 
use  bacteria  and  related  organisms  as  food.  A  culture  of  amebse 
must  provide  for  the  growth  of  bacteria,  but  this  growth  must  not 
be  so  luxuriant  as  to  overgrow  and  eliminate  the  amebse.  The 
agar  medium  recommended  by  Musgrave  and  Clegg  may  be  used. 
This  contains  agar,  20  gm.;  NaCl,  0.03  to  0.05  gm.;  beef-extract, 
0.3  to  0.5  gm.;  water,  1000  c.c.,  made  1  per  cent,  alkaline  to 
phenolphthalein.  Variations  in  the  materials  used  are  sometimes 
necessary  for  some  specialized  saprozoiites.  This  medium  is 
melted,  poured  into  a  sterile  Petri  dish,  and  allowed  to  solidify. 
The  surface  of  the  medium  is  streaked  with  the  material  containing 
the  amebae.  The  bacteria  and  amebae  will  usually  both  multiply 
if  the  plate  be  kept  at  a  suitable  temperature.  Two  operations  are 
necessary  to  secure  a  culture  in  which  it  is  known  that  all  of  the 
amebse  are  of  one  species  and  all  of  the  bacteria  of  one  kind.  The 
mixed  culture  is  placed  upon  the  medium  in  the  center  of  the 
Petri  dish.  Concentric  circles  of  the  organism  with  which  it  is 
desired  to  grow  the  ameba  are  placed  about  this.  The  amebae, 
as  they  crawl  through  the  successive  circles,  gradually  lose  the 
original  organisms  with  which  they  started  and  come  to  feed  on  the 
one  kind.  After  one  or  more  transfers  a  growth  of  the  amebae 
may  be  secured  with  the  one  species  of  organism  desired.  It  is  not 
always  possible  to  secure  growth  with  every  species  of  organism, 
as  the  different  amebae  have  been  found  to  require  various  species 
of  bacteria. 

In  order  to  secure  a  culture  containing  a  single  kind  of  ameba, 
it  is  necessary  to  isolate  a  single  individual.  Ordinarily  this  may 
be  accomplished  by  examination  of  the  surface  of  an  agar  culture 
until  an  isolated  ameba  is  found,  and  this  is  then  transferred  to  a 

27 


418  VETERINARY    BACTERIOLOGY 

fresh  plate.  Musgrave  and  Clegg  recommend  running  the  tip  of  the 
lens  against  such  an  organism  and  removing  it,  attached  to  the 
lens,  and  inoculating  the  new  medium  by  running  the  lens  down 
in  contact  with  it. 

Entamoeba  coli 

Synonym. — Amoeba  coli. 

Disease  Produced. — The  organism  is  probably  non-pathogenic. 

The  Entamceba  coli  was  probably  seen  and  recognized  by  Lambl 
in  1860.  Since  that  time  it  has  been  repeatedly  noted  by  many 
investigators.  Schaudinn,  in  1905,  first  clearly  differentiated 
the  organism  and  traced  its  life  history.  His  work  has  been  con- 
firmed and  extended  by  Craig  in  the  United  States.  The  organ- 
ism is  present  in  a  large  percentage  (50  per  cent.,  according  to 
Schaudinn)  of  healthy  individuals.  It  is  most  easily  recognized 
in  the  feces  after  the  administration  of  a  saline  cathartic. 


F  2 

Fig.  186. — Entamceba  coli:  A,  Non-motile  form;  B,  cell  showing  pseudopodia ; 
D,  Et  stages  in  cell  division;  F,  G,  H,  7,  «/,  stages  in  encystment  and  sporula- 
tion  (adapted  from  Craig). 

Morphology. — The  E.  coli  is  a  mass  of  protoplasm  not  possess- 
ing a  definite  cell-wall,  but  with  a  nucleus  containing  usually  one 
or  more  nucleoli.  One  (rarely  more)  non-contractile  vacuole  is 
occasionally  present.  The  organism  varies  from  8  to  50  //  in 
diameter,  but  in  the  majority  of  cases  is  between  10  and  20  u. 
When  encysted,  it  is  usually  between  10  and  15  u.  The  organism  is 
approximately  spherical  when  not  in  motion.  It  is  sluggishly 
motile,  and  usually  is  not  moving  when  observed  in  a  hun^ini; 
drop.  In  this  it  is  of  a  dull  gray  color.  The  protoplasm  cannot 
be  differentiated  into  endoplasm  and  ectoplasm  when  the  organ- 
ism is  at  rest.  When  in  motion,  the  ectoplasm  may  sometimes  be 
seen.  This  fact  is  of  considerable  diagnostic  value.  The  endo- 


PATHOGENIC  PROTOZOA  OF  THE  CLASS  SAKCODINA    419 

plasm  is  finely  granular,  and  rarely  contains  more  than  a  single 
vacuole.  The  nucleus  is  definite,  spherical,  and  contains  chroma- 
tin  granules  and  one  or  more  nucleoli;  it  is  5  to  8  ft  in  diameter. 
When  stained  by  Wright's  method,  the  ectoplasm  and  endoplasm 
may  be  easily  differentiated.  The  ectoplasm  stains  blue,  the 
endoplasm  violet,  and  the  nucleus  red. 

Multiplication  is  commonly  accomplished  in  the  intestines 
by  simple  division.  The  nucleus  divides  and  the  protoplasm  con- 
stricts to  form  two  individuals.  This  process  is  common  in  the 
liquid  stools,  but,  when  drier  and  firmer,  cystic  reproduction  is 
more  common.  A  refractive  hyaline  cyst  forms  about  the  spher- 
ical organism,  and  thickens.  The  protoplasm  becomes  homo- 
geneous in  appearance  and  clearer  than  before.  The  nucleus  then 
undergoes  complicated  divisions  and  recombinations,  which  ul- 
timately result  in  the  formation  of  eight  nuclei.  When  the  wall 
is  ruptured  these  nuclei,  with  bits  of  the  protoplasm,  escape  and 
constitute  eight  young  amebse. 

Pathogenesis. — Repeated  efforts  to  produce  disease  by  injec- 
tion of  Entamceba  coli  into  the  colon  of  cats  and  other  animals  have 
failed.  It  must  be  regarded  as  a  normal  inhabitant  of  the  intes- 
tines of  man.  Similar  organisms  have  been  found  in  the  intes- 
tines of  animals. 

Bacteriological  Diagnosis. — Entamceba  coli  may  be  recognized 
in  the  feces  by  a  direct  microscopic  examination  as  an  ameba 
showing  very  slight  motility,  rounded  pseudopodia,  little  or  no 
observable  differentiation  between  ectoplasm  and  endoplasm, 
comparative  lack  of  vacuoles  in  protoplasm,  and  by  the  production 
of  eight  daughter-cells  from  each  cyst. 

Entamceba  histolytica 

Synonym. — Amoeba  dysenteries. 

Disease  Produced. — Amebic  dysentery  in  man. 

Loesch,  in  1875,  described  Amoeba  coli  as  the  cause  of  a  dysen- 
tery in  man,  and  claimed  that  the  rectal  injection  of  feces  con- 
taining the  organisms  into  dogs  produced  dysentery.  Robert 
Koch,  in  1883,  showed  the  organism  to  be  present  in  the  ulcerations 
of  the  intestines.  Kartulis  firmly  established  a  probable  etio- 
logical  relationship  by  his  studies  in  Egypt,  published  in  1886. 


420 


VETERINARY   BACTERIOLOGY 


Councilman  and  Lafleur  studied  the  pathology  of  the  disease  in 
great  detail.  Schaudinri  gave  to  the  organism  its  present  name  of 
E.  histolytica,  and  described  its  morphology  with  accuracy. 

The  disease  has  been  reported  from  all  sections  of  the  world. 
It  seems  to  be  much  more  prevalent  in  tropical  than  in  temperate 
climates,  but  -has  been  identified  repeatedly  in  the  United 
States. 

Morphology. — According  to  Craig,  the  morphology  of  this 
organism  varies  so  greatly  in  culture-media  that  only  the  forms 
found  in  the  feces  can  be  regarded  as  typical.  The  organism  under 


V,,a. 


4. 


6. 


Fig.  187. — Entamceba  histolytica:  1,  Organism  in  motion — a,  Vacuoles;  b, 
red  blood-cells;  c,  pseudopodium  composed  chiefly  of  ectoplasm.  2.  Organ- 
ism showing  nucleus  a,  and  vacuoles,  6.  3.  Preliminary  changes  upon  begin- 
ning sporulation — a,  Vacuoles;  b,  chromatin  masses  escaping  from  the  nucleus 
and  scattering  through  the  cell.  4,  Chromatin  masses  scattered  through  the 
cell,  b;  c,  vacuole.  5,  Cell  budding  off  spores,  the  chromatin  masses  or  new 
nuclei  passing  through  the  ectoplasm  and  escaping  as  spores.  6,  Free  spores 
(adapted  from  Craig). 

these  conditions  varies  in  diameter  to  50  (i  or  more,  showing  it 
to  be  larger  than  E.  coli.  At  rest,  the  organism  is  spherical  or 
ovoid;  when  in  motion,  its  shape  is  extremely  variable.  It  is 
usually  actively  motile,  throwing  out  pseudopodia  much  more 
rapidly  than  E.  coli. 

Color  is  absent,  and  the  organism  appears  clear,  or  there  may 
be  a  greenish  tinge,  due  to  the  presence  of  hemoglobin  from  the 
blood  in  the  feces  and  the  blood-corpuscles  engulfed.  In  the 
larger  cells,  rarely  in  the  smaller,  the  endoplasm  and  ectoplasm 
may  be  quite  sharply  differentiated.  The  latter  is  hyaline, 


PATHOGENIC  PROTOZOA  OF  THE  CLASS  SARCODINA    421 

glasslike,  and  refractive,  much  firmer  than  the  comparatively 
delicate  membrane  of  E.  coli,  and  the  two  are  relatively  easily 
distinguishable  by  this  means.  The  ectoplasm  apparently  com- 
prises about  one-third  of  the  protoplasm.  One  or  more  vacu- 
oles  are  always  present,  as  many  as  ten  sometimes  being  found. 
The  nucleus  is  faint  and  difficult  to  distinguish  in  the  un- 
stained mount.  It  is  about  5  to  6  ft  in  diameter.  The  chro- 
matin  is  relatively  sparse.  With  Wright's  stain  the  ectoplasm 
stains  more  deeply  than  the  endoplasm,  the  opposite  of  what  is 
true  in  E.  coli. 

Reproduction  is  accomplished  in  two  ways.  The  first  consists 
of  a  division  of  the  nucleus,  followed  by  a  constriction  of  the  cell 
to  form  two  individuals.  This  process  is  essentially  the  same  as 
that  in  E.  coli.  The  method  of  spore  formation,  gemmation,  or 
budding,  is  quite  different.  When  conditions  arise  unfavorable 
to  continued  vegetative  existence,  spores  are  produced.  The 
nucleus,  by  a  process  of  fragmentation,  throws  out  chromatin 
granules  or  chromidia,  which  gradually  collect  under  the  ecto- 
plasm, form  new  nuclei,  and  are  finally  thrown  off  from  the  ex- 
terior, together  with  some  of  the  protoplasm,  as  a  spore  or  bud. 
These  spores  are  round  or  oval,  have  a  yellowish  membrane,  and 
measure  3  to  6  fit  usually  about  4  fit  in  diameter.  The  membrane 
which  forms  about  these  spores  is  resistant  to  the  penetration  and 
action  of  stains. 

Pathogenesis. — Experimental  Evidence. — Schaudinn  dried  feces 
of  dysentery  patients  after  demonstrating  that  they  contained 
E.  histolytica,  in  the  form  of  spores  in  large  numbers,  and  that  they 
were  free  from  E.  coli.  These  were  fed  to  a  kitten,  and  death 
resulted  in  fourteen  days,  with  characteristic  ulceration  of  the 
intestinal  wall.  Similar  experiments  have  since  been  repeated 
many  times.  There  is  little  or  no  doubt  of  the  pathogenicity  of 
the  organism,  and  the  disease  produced  may  be  considered  as  a 
clinical  entity. 

Character  of  Disease  and  Lesions. — The  disease  produced  is  a 
chronic  dysentery,  marked  by  intestinal  ulceration,  and  frequently 
by  abscesses  in  the  liver.  The  presence  of  the  relatively  firm 
ectoplasm  is  believed  to  account  for  the  ability  of  the  organism 
to  force  its  way  into  the  tissues  between  the  cells. 


4  ±2  VETERINARY    BACTERIOLOGY 

Immunity. — No  method  of  immunisation  against  the  E. 
histolytica  has  been  developed. 

Bacteriological  Diagnosis. — The  E.  histolytica  may  be  recog- 
nized in  the  feces,  and  separated  from  the  E.  coli  by  its  larger  size, 
its  distinct  differentiation  into  a  hyaline  refringent  ectoplasm  and  a 
more  granular  endoplasm,  the  presence  of  more  than  one  vacuole 
usually,  the  common  presence  of  erythrocytes  in  the  protoplasm 
in  the  course  of  digestion,  by  the  less  prominent  nucleus,  and  by 
formation  of  spores  by  a  process  of  budding  with  encystment. 

Entamceba  tetragena 

Synonym. — Entamccba  africana. 

This  species  was  first  described  by  Viereck  in  dysentery  in 
Africa.  Since  that  time  the  same  organism  has  been  noted  by 
Hartmann,  Werner,  and  others.  It  has  been  found  capable  of 
producing  dysentery  in  cats.  It  resembles  morphologically  the 
Entamceba  coli  more  closely  than  the  E.  histolytica,  but  differs 
by  the  formation  of  four  spores  instead  of  eight  within  the  cysts. 


CHAPTER   XLI 
PATHOGENIC  PROTOZOA  OF   THE  MASTIGOPHORA 

(Exclusive  of  the  Spirochetes) 

THE  pathogenic  forms  of  the  class  Mastigophora  differ  from 
the  preceding  in  that  they  do  not  possess  pseudopodia.  In  most 
cases  the  organism  has  a  relatively  definite  form.  The  cells  are 
motile  by  means  of  one  or  many  flagella. 

This  class  contains  many  hundreds  of  species  distributed 
among  many  genera  and  families.  Most  of  these  are  non-parasitic. 
Many  species  are  commensals  in  the  intestines  of  man  and  animals, 
and  have  been  suspected  of  producing  intestinal  disorders. 

The  protozoan  genera,  Spirocha?ta,  Treponoma,  and  Spiro- 
schaudinnia,  probably  belong  with  the  Mastigophora,  and  show 
some  distinct  resemblances  to  the  genus  Trypanosoma.  Their 
position  among  the  protozoa,  however,  is  challenged  by  many 
bacteriologists.  They  are,  therefore,  treated  as  a  separate  group 
in  the  succeeding  chapter. 

(1)  Cell-bodies  a  flexuous,  narrow  spiral  ......  Spiroch&ta  (Chapter  XLII). 

(2)  Cell-bodies  not  as  (1)  : 

An    undulating    membrane   and  terminal  flagellum 
present  .........................................  Trypanosoma. 

Undulating  membrane  not  present  .................  Herpetomonas. 


THE  GENUS  TRYPANOSOMA 

The  first  recorded  observations  of  trypanosomes  are  those  of 
Valentine,  who  saw  -them  in  the  blood  of  a  salmon  in  1841. 
Numerous  observers  found  similar  organisms  in  the  blood  of  other 
fish,  reptiles,  and  batrachia.  Lewis,  in  1878,  observed  the  first 
trypanosome  of  mammalian  blood  in  the  rat.  Evans,  in  1880, 
noted  them  in  the  blood  of  animals  infected  with  surra,  and  be- 
lieved them  to  be  the  cause  of  the  disease.  Bruce,  in  1894,  de- 

423 


424  VETERINARY   BACTERIOLOGY 

scribed  another  form  from  Africa  as  the  cause  of  nagana  or  tsetse- 
fly  disease.  Since  that  time  the  number  of  known  species  has  in- 
creased rapidly.  A  small  proportion  only  of  those  described  are 
known  to  be  pathogenic.  Among  the  pathogenic  forms,  however, 
are  to  be  found  those  that  are  among  the  most  serious  hindrances 
to  the  development  of  a  live-stock  industry,  and  even  to  human 
habitation  in  certain  countries. 

Morphology. — Trypanosomes  usually  are  elongated  cells,  more 
or  less  spindle-shaped,  rarely  almost  as  broad  as  long,  but  always 
tapering  more  or  less  to  the  ends.  Most  of  the  pathogenic  forms 
that  have  been  described  are  several  times  as  long  as  broad  when 
observed  in  the  blood.  The  anterior  end  of  the  cell  is  tipped  with  a 


Fig.  188. — Trypanosoma  equiperdum,  morphology  of  the  try panosome :  1, 
A  rather  thick,  short  trypanosome — a,  Blepharoplast ;  b,  protoplasm  (cyto- 
plasm); c,  nucleus;  d,  undulating  membrane;  e,  flagellum  attached  to  the  edge 
of  the  membrane;  /,  anterior  extension  of  the  flagellum.  2,  A  longer  cell. 
3,  4,  Trypanosomes  in  process  of  longitudinal  division  (adapted  from  Gonder 
and  Sieber). 

flagellum.  Along  one  side,  extending  longitudinally  on  the  cell, 
is  a  thin  membrane.  The  flagellum  extends  along  this  membrane 
and  forms  its  outer  edge.  The  flagellum  finally  terminates  in 
the  cell-protoplasm  near  a  granule  which  stains  deeply.  This 
granule  may  be  situated  in  various  parts  of  the  cell,  but  is  usually 
in  the  posterior  portion.  Its  relative  position  is  one  of  the  charac- 
ters used  in  the  differentiation  of  species.  It  has  been  called  by 
various  names,  as  micronucleus,  kinetonucleus,  motor  nucleus, 
centrosome,  and  blepharoplast.  This  last  name  is  to  be  preferred, 
as  it  is  the  one  that  is  most  frequently  used  in  the  descriptions,  and 
is  commonly  used  for  similar  structures  found  in  many  other 
flagellated  protozoa.  The  flagellum  is,  in  part,  therefore,  em- 


PATHOGENIC   PROTOZOA   OF  THE   MASTIGOPHORA  425 

bedded  in  the  protoplasm,  in  part  is  attached  to  the  edge  of  the 
undulating  membrane,  and  in  part  is  free  at  the  anterior  end. 
A  nucleus,  usually  situated  near  the  center  or  anterior  end,  may  be 
demonstrated  in  stained  preparations.  It  is  relatively  large  and 
granular  in  structure.  The  entire  body  of  the  trypanosome  is 
mobile,  and  the  organism  may  vary  its  shape  to  some  degree. 
It  swims  about  with  the  flagellum  in  front. 

Multiplication  is  accomplished  by  a  preliminary  division 
of  the  blepharoplast,  followed  by  that  of  the  nucleus,  and  this 
by  a  longitudinal  splitting  of  the  cell  to  form  two  individuals. 
In  cultures  the  cells  may  frequently  be  observed  in  the  form  of 
rosettes  or  clusters,  with  the  flagellar  ends  pointing  out.  These 
rosettes  do  not  occur  in  the  blood.  Transverse  division  does  not 
occur.  No  conjugation  or  fertilization  process  in  trypanosomes 
has  been  certainly  detected. 

Some  investigators  have  believed  that  trypanosomes  in  the 
animal  body  may  have  an  ultramicroscopic  stage  in  their  develop- 
ment. Bruce  and  Batemen,  in  a  series  of  careful  experiments, 
have  shown  that  this  does  not  occur. 

The  existence  of  a  regular  cycle  of  changes  in  the  life  of  a 
trypanosome  is  at  present  a  somewhat  mooted  question.  Some 
investigators  believe  that  they  have  established  the  existence 
of  a  relatively  complex  life  cycle.  Kleine,  Bruce,  and  others 
believe  that  certain  species  of  insects  which  transfer  the  disease 
must  be  considered  true  hosts,  and  that  they  do  not  become  infec- 
tive for  some  days — in  the  case  of  Tr.  gambiense  about  twenty- 
after  biting  an  infected  individual.  Rodenwalt  and  others  have 
found  that  certain  developmental  changes  take  place  in  the  gut  of 
the  insect;  others  have  failed  to  find  them.  At  present  it  may  be 
concluded  that  the  occurrence  of  developmental  changes  has  not 
been  satisfactorily  demonstrated,  although  there  is  good  reason  to 
believe  that  such  may  occur.  Carimi,  Schaudinn,  and  others  have 
recognized  what  they  believe  to  be  an  endoglobular  stage  in  the 
development  of  the  organism  in  the  body.  Battaglio  claims  to 
have  demonstrated  for  Tr.  brucei,  Tr.  lewisi,  and  Tr.  vespertilionis, 
a  developmental  cycle  in  the  blood,  which  includes  sporulation 
of  micro-  and  macrogametocytes,  with  formation  of  micro-  and 
macrogametes.  These  conclusions  have  been  combated  by  others. 


4'JO  VETERINARY   BACTERIOLOGY 

Not  all  trypanosome  infections  are  transmitted  through  insects. 
Certain  transformations  have  been  noted  with  some  forms  in  the 
body  itself.  There  is  no  evidence  that  an  insect  can  transfer  the 
organism  to  its  progeny;  that  is,  hereditary  transmission  through 
insects  plays  no  part  in  the  life-history.  This  is  in  marked  con- 
trast to  certain  other  diseases,  such  as  the  piroplasmoses. 

Cultivation  of  Trypanosomes. — Novy  and  MacNeal,  in  1903, 
gave  an  account  of  a  method  which  they  had  successfully  used  in 
the  cultivation  of  trypanosomes  in  artificial  media.  Equal  parts  of 
defibrinated  rabbit  blood  and  melted  nutrient  agar  are  mixed,  and 
the  tubes  are  slanted  and  allowed  to  solidify.  The  water  of  con- 
densation is  inoculated  with  a  small  amount  of  blood  containing 
trypanosomes.  The  first  culture  of  trypanosomes  frequently 
develops  slowly,  but  subsequent  transfers  more  quickly.  Not  all 
trypanosomes  can  be  cultivated  in  this  manner  with  equal  facility. 

Method  of  Disease  Production. — The  organisms  are  found 
typically  in  the  circulating  blood,  and  to  a  less  degree  in  the  other 
body-fluids.  Anemia  and  emaciation  are  frequently  associated 
with  the  various  trypanosome  infections.  The  spleen  is  quite 
commonly  enlarged. 

Examination  and  Staining  Methods. — Usually  the  examination 
under  a  cover-glass  of  a  drop  of  blood  from  an  infected  animal  will 
reveal  the  organism,  particularly  if  made  directly  with  the  low- 
power  objective  of  the  microscope.  The  active  motion  of  the 
organisms  reveals  their  presence  by  movements  of  the  blood-cells, 
and  they  may  be  thus  located  and  then  studied  under  the  higher 
powers.  Centrifugation  of  blood  or  body-fluids  must  be  resorted 
to  in  some  instances  to  concentrate  the  cells  when  they  are  present 
in  small  numbers  only.  Blood-films  stained  by  Wright's  method 
yield  very  satisfactory  results. 

Trypanosoma  equiperdum 

Synonym. — Tr.  rougeti. 

Disease  Produced. — Dourine:  maladie  du  coit  in  horses  (horse 
syphilis). 

Rouget,  in  1896,  first  described  this  trypanosome.  Other 
investigators  have  conclusively  established  its  etiologic  relation- 
ship to  the  disease.  It  is  of  particular  int<  r«  -t  M  the  only  try- 


PATHOGENIC    PROTOZOA    OF   THE    MASTIGOPHORA  427 

panosome  disease  of  importance  in  Europe  and  in  North  Amer- 
ica. 

Distribution. — The  disease  is  known  from  Germany,  Austria, 
France,  and  southern  European  countries,  northern  Africa,  western 
Asia  and  India,  Chile,  Java,  and  several  local  epidemics  have 
occurred  in  North  America  (Illinois,  Nebraska,  Wyoming,  South 
Dakota,  and  northwestern  Canada). 

Morphology  (See  Fig.  188). — Moore  and  Bredini,  in  a  study  of 
this  trypanosome  as  it  occurs  in  artificially  infected  rats,  concluded 
that  it  passed  through  certain  developmental  stages,  finally  being 
converted  into  rounded  bodies  with  two  long  delicate  flagella. 
The  organism,  as  it  occurs  in  the  lesions  and  blood  of  the  horse,  is 
a  slender  cell,  usually  about  25  to  28  //  in  length.  There  are  no 
particular  differentiating  characters  between  this  organism  and  the 
ones  associated  with  other  trypanosomiases  of  the  horse.  The 
blepharoplast  is  distinct,  the  membrane  considerably  folded,  the 
nucleus  central,  and  the  free  flagellum  about  ^  to  ^  the  length  of 
the  organism.  Protoplasmic  granules  are  never  present. 

Cultivation. — Thomas  and  Breinl  have  succeeded  in  cultivating 
the  organism  in  a  modified  Novy  and  MacNeal  medium  in  one 
trial  out  of  nineteen. 

Pathogenesis. — Experimental  Evidence. — The  disease  may  be 
transmitted  experimentally  to  the  horse,  the  ass,  to  fowls,  and  even 
to  ruminants  and  apes,  according  to  some  observers.  The  disease 
occurs  naturally  only  among  the  equines. 

Character  of  Disease. — The  infected  animal  becomes  emaciated, 
while  whitish  or  chalk-like  areas  appear  in  the  skin  and  mucosa  of 
the  external  genitalia.  Ulcers  frequently  develop,  particularly 
upon  the  penis.  The  disease  usually  runs  a  chronic  course.  The 
animal  frequently  becomes  gradually  paralyzed.  Recovery  is  in- 
frequent . 

Immunity. — Animals  that  recover  from  the  disease  are  thereby 
rendered  immune  to  a  second  infection.  They  are  not,  however, 
rendered  immune  to  infection  with  another  trypanosome,  such  as 
that  of  surra.  It  appears  that  an  immunity  acquired  during 
pregnancy  may  be  transmitted  to  the  offspring.  No  method  of 
immunization  based  upon  the  organism  or  its  products  has  been 
developed. 


428  VETERINARY   BACTERIOLOGY 

Bacteriological  Diagnosis. — This  may  occasionally  be  accom- 
plished by  a  microscopic  examination  of  the  fluids  from  the  lesions 
and  the  identification  of  the  characteristic  trypanosome  in  stained 
mounts.  The  organisms  can  rarely  be  found  in  the  blood  of  the 
general  circulation.  The  blood-tinged  fluid  secured  from  the 
plaques  when  they  first  appear  is  the  most  favorable  material 
for  their  identification.  They  may  be  found  in  the  blood-stream 
of  artificially  infected  laboratory  animals. 

Transmission. — The  disease  is  commonly  transmitted  from  one 
animal  to  another  through  coition.  Whether  or  not  the  organism 
can  enter  through  the  intact  mucosa  is  not  certainly  known,  but 
appears  probable.  Blood  containing  the  organism  will  infect 
a  rabbit  if  placed  in  the  conjunctival  sac.  Sieber  and  Gonder 
claim  to  have  succeeded  in  transmitting  the  disease  through  the 
medium  of  the  fly  Stomoxys  caltitrans,  but  this  certainly  is  not  the 
common  method.  No  developmental  stages  could  be  observed 
in  the  fly. 

Trypanosoma  evansi 

Synonym. — Spirochoeta  evansi. 

Disease  Produced. — Surra  in  horses,  cattle,  carabou  or  water 
buffalo,  camels,  dogs,  goats,  and  sheep. 

The  disease  has  been  known  for  a  long  time  from  southern  Asia. 
The  organism  was  first  described  from  India  by  Evans  in  1880. 

Distribution. — The  disease  is  known  from  India,  China,  the 
Philippines,  Africa,  and  Australia. 

Morphology. — The  organism  as  it  occurs  in  the  blood  is  actively 
motile.  It  is  usually  between  20  and  30  [*>  in  length,  and  from  1  to 
2  ^  in  diameter.  It  tapers  to  the  anterior  end,  but  the  posterior 
is  somewhat  blunt.  The  undulating  membrane  and  the  free 
flagellum  are  well  differentiated.  This  organism  can  scarcely 
be  separated  from  Tr.  brucei  on  the  basis  of  morphology. 

Cultivation. — This  organism  has  been  cultivated  on  Novy  and 
MacNeal's  medium,  but  only  after  repeated  trials. 

Pathogenesis. — The  disease  may  be  readily  transmitted  to 
susceptible  animals  by  the  injection  of  blood  containing  the 
trypanosome.  The  disease  itself  is  characterized  in  the  horse  as  a 
relapsing  fever,  with  eruptions,  either  generalized  or  localized 
in  the  skin.  Petechial  hemorrhages  of  the  mucosae  are  frequent. 


PATHOGENIC   PROTOZOA   OF  THE   MASTIGOPHORA  429 

The  subcutaneous  tissues  are  infiltrated  and  edematous.  It  is 
practically  invariably  fatal.  Cattle  are  relatively  resistant  to  the 
disease,  and  in  these  animals  recovery  usually  occurs,  but  the  blood 
may  remain  infective  for  a  considerable  period.  Buffalo  frequently 
succumb.  Camels,  elephants,  and  dogs  are  not  infrequently 
infected. 

This  disease  resembles  nagana  clinically,  and  the  causal  organ- 
isms can  scarcely  be  differentiated.  Animals  immunized  against 
the  one  disease  are  susceptible  to  the  other,  which  would  seem  to 
establish  specific  differences  sufficient  to  separate  the  organisms 
as  distinct  species. 

Bacteriological  Diagnosis. — The  organisms  gradually  increase  in 
numbers  in  the  blood  during  the  onset  of  the  disease,  and  have  been 
found  as  numerous  as  350,000  per  cubic  millimeter.  They  are 
frequently  not  present  in  the  blood  between  the  periods  of  fever. 

Transmission.— Fraser  and  Symons  state  that  in  the  Federated 
Malay  States  four  species  of  the  fly  genus  Tabanus,  particularly 
T.  fumifer,  are  responsible  for  the  spread  of  the  disease.  Probably 
other  flies  may  carry  the  organism  as  well.  Experiments  seem  to 
show  that  the  transference  in  this  case  is  merely  mechanical,  and 
that  there  is  no  developmental  cycle  in  the  intermediate  host. 
Carnivorous  animals  may  be  infected  by  ingestion  if  there  are 
lesions  in  the  mucous  membranes. 

Trypanosoma  brucei 

Disease  Produced. — Nagana  or  tsetse-fly  disease  in  horses, 
cattle,  camels,  buffalo,  antelopes,  and  related  wild  animals,  pos- 
sibly the  elephant. 

The  fact  that  this  disease  follows  the  bite  of  the  tsetse  fly  has 
long  been  known  by  African  natives,  and  the  early  explorers  con- 
firmed their  belief.  Bruce,  in  1896,  described  the  trypanosome 
which  causes  the  disease. 

Distribution. — Known  only  in  Africa,  particularly  in  Zulu  land. 

Morphology. — The  organism  is  sluggishly  motile.  It  is  usually 
between  25  and  30  u  in  length  and  1.5  to  2.5  (i  in  width.  Granules 
may  generally  be  observed  in  the  protoplasm.  Irregular  forms 
occur  in  the  blood  after  death,  and  in  the  lymphatic  glands,  spleen, 
bone-marrow,  liver,  and  lungs  during  life.  Kleine  believes,  from 


430  VETERINARY    BACTERIOLOGY 

his  experiments  with  fly  transmission,  that  there  must  be  a  develop- 
mental stage  occurring  in  the  insect. 

Isolation  and  Culture. — Novy  and  MacNeal  succeeded  in  grow- 
ing the  organism  of  nagana  in  the  medium  already  described.  Only 
a  few  tubes  out  of  a  large  number  were  found  to  show  growth. 

Pathogenesis. — Experimental  Evidence. — Inoculation  of  the 
mouse,  rat,  dog,  cat,  and  monkey  results  in  an  acute  infection;  of 
the  rabbit,  guinea-pig,  equines,  and  swine,  in  a  subacute  infection; 
and  of  cattle,  goats,  and  sheep,  in  a  chronic  infection. 

Character  of  Disease  and  Lesions  Produced. — The  disease  is  of 
greatest  importance  in  the  equines.  The  incubation  period  is 
from  three  to  twelve  days  (Theiler).  -There  is  a  continued  or 
remittent  fever  and  a  watery  discharge  from  the  eyes  and  nose. 
The  animal  becomes  much  emaciated  before  death,  which  usually 


Fig.  189. — Trypanosoma  brucei  (adapted  from  Gonder  and  Sicber). 

occurs  in  from  two  weeks  to  three  months.  Edema  of  the  ventral 
region  is  common.  Hypertrophy  of  the  spleen  is  the  most  constant 
lesion.  The  lymph-glands  are  generally  enlarged. 

Immunity. — Rodet  and  Vallet  believe  that  the  organism  is 
rapidly  destroyed  in  the  spleen.  No  practicable  method  of  im- 
munizing against  the  organism  has  been  developed.  It  has  been 
found  that  the  injection  of  human  serum  into  laboratory 
animals  at  intervals  will  greatly  prolong  their  life,  but  will  not 
cure.  There  is  probably  some  relationship  between  immunity 
of  man  and  the  trypanicidal  character  of  his  serum.  Goats, 
sheep,  and  cattle  show  a  considerable  percentage  of  cures  and 
are  thereafter  immune.  Their  serum,  however,  has  little  im- 
munizing power. 

Bacteriological  Diagnosis. — 'Stained  mounts  of  the  blood  from 
infected  animals  will  generally  reveal  the  chani<-teri-iir  parasites. 


PATHOGENIC   PROTOZOA   OF   THE   MASTIGOPHOKA  431 

Centrifugation  of  the  blood  will  frequently  result  in  the  collec- 
tion 'of  the  organisms  in  a  layer  just  at  the  surface  of  the  cor- 
puscles. 

Transmission. — The  disease  is  commonly  transmitted  from  one 
animal  to  another  by  the  bite  of  the  tsetse  fly  (Glossinia  mwsitanx). 
The  herbivorous  animals  native  to  sections  of  the  country  where  the 
disease  is  prevalent  are  almost  invariably  infected,  and  render 
infection  of  other  animals  easy.  Kleine  experimentally  showed 
that  another  species  of  Glossinia  (G.  palpalis)  could  transmit  the 
disease.  He  found  that  the  flies  did  not  become  infective  until 
eighteen  days  had  elapsed.  Other  experimenters  have  found  that 
the  fly  lost  its  infectiveness  within  a  day  or  two  after  feeding  upon 
an  infected  animal,  and  have  concluded  that  transmission  is  wholly 
a  mechanical  affair.  It  seems  possible  that  transmission  may 
occur  mechanically  in  this  manner,  or  there  may  be  a  true  develop- 
mental cycle  in  the  body  of  the  insect. 

Trypanosoma  equinum 

Synonym. —  Tr.  elmassiani. 

Disease  Produced. — Mai  de  caderas  of  the  horse  (Spanish 
caderas  =  rump  or  hindquarter) . 

Elmassian,  in  1901,  announced  his  discovery  of  the  specific 
trypanosome  of  this  disease  in  Paraguay.  Voges,  in  Argentina, 
confirmed  this  discovery  in  the  following  year.  The  disease  is 
so  prevalent  in  some  sections  that  cattle  are  used  exclusively  for 
riding  and  driving. 

Distribution. — Parts  of  South  America,  particularly  Brazil, 
Paraguay,  and  Argentina. 

Morphology. — This  trypanosome  resembles  those  of  surra  and 
nagana,  but  the  blepharoplast  is  so  inconspicuous  that  it  may  be 
readily  overlooked.  The  cell  is  usually  between  22  and  24  a  in 
length  and  1.5  u.  in  width.  Cells  about  to  divide  are  somewhat 
larger.  The  difference  in  the  blepharoplasts  of  this  and  most  other 
forms  renders  identification  easy  even  in  mixed  infections. 

Pathogenesis. — Experimental  Evidence. — Inoculation  of  the 
organism  causes  a  fatal  infection  in  the  horse;  the  mule  and  donkey 
are  somewhat  more  resistant,  as  are  mice,  rats,  and  other  rodents, 
rabbits,  and  other  laboratory  animals.  Birds  cannot  be  infected. 


432  VETERINARY   BACTERIOLOGY 

The  pig,  sheep,  goat,  and  ox  may  show  transitory  symptoms,  but 
are  highly  refractory. 

Character  of  Disease  and  Lesions. — The  disease  differs  from  surra 
and  nagana  in  the  almost  complete  absence  of  edema,  and  is  charac- 
terized by  a  paralysis  of  the  hindquarters.  There  is  a  progressive 
emaciation,  fever,  and  the  hindquarters  become  weak;  the  horse 
in  walking  scarcely  raises  the  hoof  above  the  ground.  Finally, 
the  animal  supports  itself  by  leaning,  or  falls  to  the  ground. 
There  are  no  lesions  upon  the  genital  organs. 

Immunity. — No  method  of  practicable  immunization  against 
the  organism  has  been  developed. 

Bacteriological  Diagnosis. — The  organism  may  be  found  in  the 
blood,  particularly  during  the  fever  paroxysms. 

Transmission. — The  disease  is  evidently  endemic  in  certain 
parts  of  South  America  in  rodents  or  other  animals.  One  of  these, 
the  capybara  (Hydrochcerus  capybara),  has  been  found  to  be  in- 
fected, and  it  is  said  that  stockmen  can  sometimes  foretell  an  out- 
break of  the  mal  de  caderas  by  the  death  of  many  of  these  animals 
in  the  vicinity.  The  method  of  transmission  is  not  certainly 
known.  Flies  have  been  supposed  to  act  as  carriers,  but  definite 
proof  is  lacking. 

Trypanosoma  dimorphon 

Diseases  Produced. — Gambian  horse  sickness,  trypanosomiasis 
in  horses  and  other  equines,  cattle,  sheep,  and  goats. 

Button  and  Todd,  in  1902,  reported  the  discovery  of  a  specific 
trypanosome  in  a  disease  of  horses  in  Senegambia.  The  same,  or 
very  similar  trypanosome,  has  since  that  time  been  reported  from 
many  African  localities  in  other  animals  as  well. 

Distribution. — Various  localities  in  Africa  (French  Guinea, 
Zanzibar,  Sierra  Leone,  Mozambique,  Zululand). 

Morphology. — The  organism  is  characterized  by  the  absence  of  a 
free  flagellum,  the  flagellum  terminating  with  the  undulating  mem- 
brane. It  is  dimorphic,  some  of  the  cells  being  20  to  25  u  in  length, 
others  only  about  12  /w.  Transitional  forms  between  these  ex- 
tremes may  be  found.  The  undulating  membrane  is  not  well 
developed.  Protoplasmic  granules  are  very  rare  or  are  absent  in 
the  cell. 

Pathogenesis. — Experimental  Evidence. — The  disease  has  been 


PATHOGENIC   PROTOZOA   OF   THE   MASTIGOPHORA  433 

experimentally  produced  by  the  inoculation  of  the  organism  into 
sheep,  cattle,  goats,  rabbits,  horses,  and  white  rats. 

Character  of  Disease  and  Lesions. — The  infection  is  acute  in  the 
rat;  acute  or  chronic  in  the  rabbit  and  dog;  and  chronic  in  the 
ox,  sheep,  and  in  equines.  It  produces  a  severe  anemia  with 
changes  in  the  red  blood-cells.  In  the  horse  there  is  progressive 
emaciation.  A  marked  edema  is  rarely  produced.  Death  does 
not  occur  usually  for  months  after  infection.  Recovery  sometimes 
occurs. 

Immunity. — No  practicable  method  of  immunization  by  means 
of  the  organism  or  its  products  has  been  developed. 

Bacteriological  Diagnosis. — The  organism  may  be  found  in  the 
blood  at  certain  periods,  but  repeated  examination  is  sometimes 
necessary.  Inoculation  of  other  animals  with  the  blood  will 
nevertheless  show  it  still  to  be  infective. 

Transmission.— Pecaud  states  that  immediate  transmission 
may  be  due  to  a  fly,  Stomoxys.  Tsetse  flies  are  found  in  the  region 
in  which  the  disease  is  found,  and  may  be  responsible  for  its  spread. 
The  exact  method  of  transmission  is  not  certainly  known. 

Trypanosoma  congolense 

Brodin  described  a  disease  in  the  Congo  Free  State  which 
closely  simulated  nagana,  but  the  trypanosome  resembled  rather 
Tr.  dimorphon.  Laveran,  as  a  result  of  inoculation  experiments, 
has  concluded  that  this  is  a  distinct  species.  It  has  been  reported 
from  several  localities  in  southern  Africa.  It  is  fatal  for  cattle 
and  sheep.  The  organisms  are  10.5  to  15.5  u  in  length  and  1.7  to 
2.5  u  wide.  The  flagellum  shows  no  free  portion,  hence  animal 
inoculations  are  necessary  to  differentiate  between  this  and  Tr. 
dimorphon. 

Trypanosoma  pccaodi 

Disease  Produced. — Baleri  or  trypanosomiasis  in  horses, 
cattle,  sheep,  and  goats. 

This  organism  was  described  by  Laveran  from  the  blood  of 
inoculated  sheep  brought  to  Paris  by  Cazalbou. 

Distribution. — The  disease  is  known  from  the  French  Sudan. 

Morphology. — The  organism  closely  resembles  Tr.  dimorphon. 

28 


434 


VETERINARY   BACTERIOLOGY 


Two  forms  are  described:  long  slender  cells,  25  to  35  {i  in  length 
by  about  1.5  ft  in  width,  with  narrow,  undulating  membrane  and 
fairly  long  flagellum,  and  short  broad  forms,  14  to  20  by  3  to  4  [i, 
with  no  free  flagellum  and  a  wide,  undulating  membrane. 


cro 


Fig.  190. — Trypanosoma  pecaudi  (Laveran). 

Pathogenesis. — Character  of  Disease  and  Lesions. — In  the  horse 
the  disease  is  characterized  by  repeated  attacks  of  a  severe  fever, 
swellings  in  various  parts  of  the  body,  injection  of  the  conjunctiva, 
and  a  considerable  degree  of  emaciation. 

Trypanosoma  cazalboui 

Disease  Produced. — Souma  or  soumaya  in  cattle,  sheep,  horses, 
and  mules. 

Distribution. — Africa,  from  the  French  Sudan,  French  Congo, 
Upper  Nile. 

Morphology. — The  organism,  including  its  free  flagellum,  is 
about  21  by  1.5  {i.  The  oval  nucleus  is  centrally  located.  The 
undulating  membrane  is  poorly  developed  and  is  little  folded.  The 
terminal  portion  of  the  flagellum  is  free.  There  are  no  marked 
characters  differentiating  the  organism  from  Tr.  evansi. 

Pathogenesis. — Experimental  Evidence. — Laboratory  animals, 
particularly  the  rodents,  seem  to  be  relatively  immune.  The 
infection  may  be  readily  transmitted  to  horses  and  cattle  and  the 
smaller  ruminants.  Cross-inoculation  experiments  have  shown 
the  disease  to  be  distinct  from  surra. 

Character  of  Disease. — It  attacks  cattle  generally.  The  disease 
leads  to  a  progressive  emaciation,  the  skin  is  harsh,  and  there  is 


PATHOGENIC   PROTOZOA   OF   THE   MASTIGOPHORA 


435 


a  staring  coat.  In  many  individuals  the  lower  surfaces  of  the 
body  show  marked  edema.  The  temperature  is  variable.  The 
disease  generally  lasts  seven  or  eight  months. 

Bacteriological  Diagnosis. — The  organisms  are  present  in  the 
blood,  usually  in  small  numbers  only. 


oo 


Fig.  191. — Trypanosoma  cazalboui  (Laveran). 

Transmission. — Pecaud  concludes  that  Glossinia  palpalis  is 
responsible  for  the  distant  transmission  of  the  organism,  and  that 
members  of  the  fly-genera  Stomoxys  and  Tabanus  may  produce 
immediate  transmission. 

Trypanosoma  theileri 

Disease  Produced. — Associated  with  galziekte  or  gall-sickness 
in  bovines. 

This  organism  was  first  noted  by  Theiler  and  was  described 
at  greater  length  by  Laveran. 

Distribution. — A  large  part  of  South  Africa,  possibly  also  in 
India. 

Morphology. — The  organism  associated  with  this  disease  is  of 
unusual  size,  30  to  70  u  in  length,  and  2  to  5  fi  in  width.  This 
alone  is  sufficient  to  differentiate  it  from  other  forms.  There 
is  a  long,  free  flagellum.  The  nucleus  is  central,  but  the  blepharo- 
plast  is  a  considerable  distance  from  the  posterior  end.  Many 
protoplasmic  granules  may  usually  be  seen. 

Pathogenesis. — The   organism   seems   to   be   inoculable   only 


436  VETERINARY   BACTERIOLOGY 

into  cattle.  It  resembles  the  rat  trypanosome  in  being  thus  limited 
to  a  single  host.  The  organism  was  first  described  as  the  cause  of 
galziekte,  but  the  recent  investigations  of  Theiler  seem  to  show  it 
to  be  a  relatively  harmless  commensal. 

Bacteriological  Diagnosis. — The  organisms  are  quite  common  in 
the  blood,  but  soon  diasappear. 

Transmission. — It  has  been  found  to  be  transmitted  by  the 
bite  of  the  fly,  Hypobosca  rufipes. 

Trypanosoma   gambiensc 

Synonyms. — Tr.  ugandense;  Tr.  castellanl. 

Disease  Produced. — Human  trypanosomiasis,  or  sleeping  sick- 
ness in  man. 

The  disease  known  as  sleeping  sickness  has  been  known  among 
the  negroes  of  the  west  coast  of  Africa  for  over  a  century.  Button, 
in  1901,  had  an  opportunity  to  examine  the  blood  of  a  European 
infected  with  the  disease,  and  found  and  described  the  causal 
trypanosome.  Other  investigators  have  abundantly  confirmed 
and  extended  his  observations. 

Distribution. — The  disease  is  endemic  upon  the  western  coast 
of  Africa  and  in  certain  of  the  central  portions. 

Morphology. — The  organism  is  17  to  28  by  1.4  to  2  [i.  Forms 
undergoing  division  are  somewhat  larger.  The  free  flagellum  may 
be  one-fourth  to  one-third  the  length  of  the  body.  Rarely  no 
free  portion  of  the  flagellum  can  be  demonstrated.  The  undulating 
membrane  is  narrow.  The  blepharoplast  is  near  the  posterior  end. 
Protoplasmic  granules  that  stain  like  chromatin  are  commonly 
observed. 

Pathogenesis. — Experimental  Evidence. — The  disease,  with  its 
characteristic  clinical  symptoms,  may  be  reproduced  by  the  injec- 
tion of  blood  containing  the  organisms  into  the  monkey.  Dogs, 
jackals,  cats,  guinea-pigs,  and  rabbits  are  readily  infected.  Mice 
frequently  recover  and  are  thereafter  immune.  Goats  and  sheep 
are  relatively  refractory,  but  sometimes  succumb.  It  may  pro- 
duce a  mild  chronic  infection  in  the  horse  and  in  cattle. 

Character  of  Disease. — The  disease  is  insidious  in  its  onset. 
Two  distinct  stages  may  be  recognized.  These  were  for  a  long  time 
supposed  to  be  different  diseases.  In  the  first  stage  the  organisms 


PATHOGENIC    PROTOZOA   OF   THE    MASTIGOPHORA  437 

appear  in  the  blood ;  there  may  or  may  not  be  fever.  In  the  second 
stage  pains  in  the  back,  tremors,  and  drowsiness  supervene. 
Finally,  the  patient  dies  in  a  comatose  condition.  In  this  second 
stage  the  organisms  are  present  in  numbers  in  the  cerebrospinal 
fluid.  The  disease  appears  to  be  always  fatal,  but  may  run  a 
chronic  course  lasting  several  years. 

Immunity. — No  practicable  method  of  immunization  has  been 
developed. 

Bacteriological  Diagnosis. — The  organisms  are  usually  scanty 
in  the  blood,  and  centrifugation  is  necessary  to  find  them.  They 
may  usually  be  demonstrated  in  the  fluid  secured  by  a  lumbar 
puncture.  They  may  also  commonly  be  demonstrated  by  punc- 
turing an  enlarged  lymphatic  gland  and  examining  the  fluid  secured. 

Transmission. — One  of  the  tsetse  flies,  Glossinia  palpalis,  has 
been  found  to  transfer  the  disease. 

Trypanosoma  critzi 

Chagas,  in  1909,  described  this  organism  as  the  cause  of  a 
disease  in  man  called  opilacao,  and  hitherto  confounded  with 
ankylostomiasis.  It  is  transmitted  by  the  bite  of  a  bug  (Conor- 
rhinus  sp.).  This  work  needs  confirmation. 

Trypanosoma  calmettei 

Mathis  and  Leger,  in  1909,  described  a  non-pathogenic  try- 
panosome  from  the  blood  of  the  domestic  fowl  in  Tonkin.  It 
occurs  but  rarely. 

Trypanosomes  in  Birds 

Trypanosomes  have  been  described  from  the  blood  of  a  great 
number  of  wild  birds.  They  are  not  known  to  be  pathogenic. 

Trypanosoma  lewisi 

Chaussat,  in  1850,  and  Lewis,  in  1877,  noted  the  presence 
of  a  flagellate  in  the  blood  of  rats.  It  is  so  commonly  present 
in  the  blood  of  rats  in  many  parts  of  the  world  that  it  has  been 
frequently  used  in  the  laboratory  for  study  and  demonstration, 
although  its  pathogenic  properties  are  almost  nil.  This  trypano- 
some  cannot  be  transmitted  to  any  other  genus  of  mammals  so  far 
as  known :  even  the  closely  related  genera  of  the  rodents  are  re- 


438  VETERINARY   BACTERIOLOGY 

fractory.  It  evidently  is  a  highly  specialized  commensal.  The 
organism,  with  its  flagellum,  measures  about  24  to  25  [i  in  length 
by  1.5  U  in  width.  Protoplasmic  granules  are  frequently  present. 

THE  GENUS  HERPETOMONAS 

This  genus  includes  certain  flagellates  that  have  an  essentially 
trypanosome-like  structure,  without  an  undulating  membrane. 
The  cell  is  elongated  in  the  typical  Herpetomonas;  the  closely 
related  genus  Crithridia  comprises  those  forms  in  which  the  body 
is  much  shortened.  The  flagellum  is  anterior,  the  blepharoplast 
distinct,  as  in  the  trypanosomes,  and  the  nucleus  centrally  located. 

Organisms  of  this  genus  have  been  frequently  reported  from 
the  gut  of  the  mosquitoes  and  flies.  Certain  of  the  trypanosomes 
sometimes  assume  shapes  that  resemble  closely  the  Herpetomonas. 
The  genus  assumes  pathogenic  significance,  principally  because 
of  the  tentative  classification  of  certain  protozoa  known  as  the 
Leishman-Donovan  bodies  as  members  of  this  genus.  Three 
species  have  been  described. 

Herpetomonas  donovani 

Synonyms. — Leishman-Donovan  bodies;  Leishmania  dono- 
vani; Trypanosoma  donavani. 

Disease  Produced. — Kala-azar,  cachexial  or  Dumdum  fever 
in  man. 

Leishman,  in  1900,  observed  this  parasite  in  smears  from 
the  spleen  of  a  patient  that  had  died  of  Dumdum  fever.  His 
account  was  published  in  1903. 

Distribution. — Throughout  southern  Asia  and  northern  Africa. 

Morphology. — The  organism  as  it  occurs  in  the  body  is  com- 
monly intracellular.  It  is  found  principally  in  the  spleen,  liver, 
bone-marrow,  and  lymph-glands.  It  is  oval,  spherical,  or  pear- 
shaped,  usually  between  2  u  and  3.5  ^  in  length  and  1.5  to  2  fi  in 
width.  Two  staining  granules  occur  in  the  interior,  the  larger 
spherical,  and  the  smaller,  and  more  deeply  staining  granule, 
somewhat  elongated.  The  organisms  multiply  by  a  preliminary 
division  of  both  of  the  chromatic  granules  (nucleus  and  blepharo- 
plast), followed  by  a  constriction  of  the  cell.  In  cultures  typical 
flagellates  are  produced.  The  organism  elongates  somewhat, 


PATHOGENIC    PROTOZOA   OF   THE   MASTIGOPHORA 


439 


and  a  vacuole  appears  at  one  side  of  the  blepharoplast,  and  from 
this  a  single  flagellum  develops.  The  body  finally  assumes  an 
elongated  form  not  unlike  a  trypanosome,  without  an  undulating 
membrane.  This  is  the  Herpetomonas  stage.  This  may  now 
divide  longitudinally,  frequently  unequally,  splitting  off  very 
slender  cells.  The  systematic  position  of  this  organism  is  still 
somewhat  in  doubt.  It  may  be  that  it  should  be  regarded  as 
belonging  to  a  distinct  genus,  and  the  name  Leishmania  used 
instead  of  Herpetomonas. 

Pathogenesis. — The  disease  is  characterized  by  enlargement 
of  the  spleen  and  by  fever. 

Transmission. — It  is  believed  that  the  parasite  is  transferred 
from  the  dog  to  man  through  some  intermediate  host. 

Leishmania  (Herpetomonas  ?)  infantum 

Xicolle  has  described  an  organism  similar  to  the  preceding 
from  a  disease  which  he  calls  infantile  kala-azar.  The  organisms 


Fig.  192. — Herpetomonas  infantum:  A,  Organisms  from  the  spleen  of  a  child; 
B,  a  mononuclear  containing  the  organisms,  and  C,  an  endothelial  cell  from  the 
spleen;  D,  various  stages  in  the  development  of  the  Herpetomonas  form  from 
the  Leishman-Donovan  bodies  (Nicolle). 

resemble  the  preceding,  but  are  believed  to  constitute  a  separate 
species.  The  disease  is  primarily  one  of  dogs,  which  may  be 
transmitted  to  children. 

Leishmania  tropica 

Synonyms. — Ovoplasma  orientale:  Helcosoma  tropicum. 

Wright  has  described  a  similar  organism  as  the  cause  of  oriental 
sore  or  Delhi  boil  in  man.  It  is  probably  transmitted  likewise  by 
some  biting  insect.  The  dog  may  be  infected  and  show  clinical 
symptoms  similar  to  man. 


CHAPTER  XLII 

SPIROCHETE  GROUP 

THERE  is  probably  more  confusion  relative  to  the  classification 
of  the  members  of  this  group  of  organisms  than  in  any  other 
group  of  bacteria  or  protozoa.  In  the  first  place,  there  is  by 
no  means  an  agreement  among  investigators  as  to  whether 
these  organisms  should  be  included  under  the  heading  of  bacteria 
or  of  protozoa.  There  seems  to  be  more  evidence  in  recent 
literature  of  protozoan  rather  than  of  bacterial  relationships. 
Second,  it  is  evident  that  the  group  is  not  homogeneous,  and 
efforts  have  been  made  to  separate  the  group  into  several  genera. 


Fig.  193. — Spirochceta  pinnce:  A,  B,  Cells  showing  the  undulating  membrane; 
C,  a  coiled  organism  (adapted  from  Gonder) . 

In  not  a  single  case  have  we  a  full  and  satisfactory  knowledge  of  the 
life  history,  particularly  in  those  forms  which  are  transmitted  from 
one  animal  to  another  by  parasites.  Until  this  is  worked  out  it 
will  be  impossible  to  make  a  separation  of  the  different  types  into 
genera  on  the  basis  of  true  relationships.  In  the  discussion  of  the 
group  the  genus  name  of  Spirochoeta  is  retained,  the  first  of  the 
synonyms  given  being  the  one  which  has  been  suggested  by  Blanch- 
ard  in  his  revision  of  the  group  on  the  basis  of  protozoan  relation- 
ship* 

The  argument  advanced  for  protozoan  relationships  may  be 

440 


SPIROCHETE   GROUP  441 

summarized  as  follows.  According  to  several  observers,  multipli- 
cation, frequently,  though  not  invariably,  takes  place  by  longitu- 
dinal rather  than  transverse  division  of  the  cell.  Morphologic- 
ally, it  is  believed  that  these  organisms  differ  from  the  true  bacteria 
by  being  in  all  cases  flexible,  and  swimming  with  a  sinuous  motion. 
In  several  species,  undulating  membranes  similar  to  those  of  the 
protozoa  have  been  demonstrated.  Chromidia-like  bodies,  re- 
sembling the  scattered  nuclei  in  some  protozoa,  have  been  identified. 

Sp.  dentium, 

Sp.  insequalis 


Sp.  tenuis 


Sp.  denticola 


Sp.  undulata'  ^H  ^Sp.  dentium 


Sp  recta 

Fig.  194. — Spirochaetae  of  different  species.     From  a  smear  from  a  tonsillar 
lacuna  stained  by  Burril's  India  ink  method  (Gerber) . 

According  to  Prowazek,  plasmolysis  does  not  take  place  with  salt 
solutions  several  times  as  concentrated  as  those  required  for 
plasmolysis  of  bacterial  cells.  Certain  spirochetes  have  been 
observed  to  enter  red  blood-cells,  and  there  assume  a  coiled  condi- 
tion. A  multiplication  of  the  organisms  has  been  observed  in 
the  eggs  of  a  tick  which  transmitted  a  spirochete  disease,  as  have 
also  certain  forms  which  have  been  interpreted  as  stages  in  a  more 
complex  life-history.  Leishman  has  found  that  certain  spiro- 
chetes, when  ingested  by  the  tick,  lose  their  motility  and  change 


442  VETERINARY   BACTERIOLOGY 

morphologically,  with  resultant  liberation  of  small  bodies  which 
stain  like  chromatin.  These  are  of  various  shapes — rods,  cocci, 
or  spirals.  They  enter  the  lining  cells  of  the  malpighian  tubules; 
they  are  found  in  the  oviduct,  the  ovary,  and  the  immature  eggs, 
and  in  all  stages  of  development  of  the  young  tick.  Inoculation  of 
material  containing  these  bodies,  but  not  true  spirochetes,  resulted 
in  the  development  of  tick-fever.  The  details  of  this  life-history 
are  in  present  need  of  elucidation.  It  has  been  claimed  by  Mar- 
choix  that  the  virulence  of  the  spirochete  of  fowl  septicemia  can 
be  preserved  only  by  passing  through  the  body  of  the  tick  which 

transmits  the  disease.    Con- 
tinual transfer  of  the  organ- 
^  ism     from     one     fowl     to 

another,  without  the  inter- 
mediation of  the  tick,  causes 
a  gradual  decrease  in  viru- 
lence. This  would  seem 
to  indicate  that  a  part  of 
the  life  cycle  of  this  organ- 
ism must  be  passed  in  the 
body  of  the  intermediate 
host  or  tick.  None  of  this 
group  of  organisms  may  be 

,-,.  cultivated  uponthe  common 

.frig.  195. — Spirochceta  obermeieri  show- 
ing flagella  distributed  over  the  body  of     laboratory  media,  and  they 
the  organism  (Frankel).  can  be  induced  to  multiply 

in    vitro    only    under  very 

special  conditions.  All  these  facts  would  seem  to  mal^e  out  a 
strong  case  for  those  who  believe  in  the  protozoan  relationships. 

There  are,  on  the  other  hand,  investigators  who  believe  quite 
as  firmly  that  these  organisms  are  bacteria.  Novy  and  Knapp 
studied  with  great  care  a  spirochete  of  relapsing  fever.  They 
concluded  that  division  was  always  transverse,  and  that  the 
longitudinal  divisions  reported  by  others  were  accidental  associa- 
tions or  intertwining  of  two  organisms.  Their  results  with  the 
use  of  plasmolyzing  agents  received  an  interpretation  quite  the 
reverse  of  other  investigators.  Experiments  in  immunization, 
the  resistance  to  heat,  stability  of  form,  and  staining  qualities  of 


SPIROCHETE   GROUP  443 

the  flagellum  all  associated  the  organism  with  the  bacteria.  Bonel, 
Frankel,  and  others  claim  to  have  demonstrated  the  presence  of 
numerous  peritrichic  flagella  on  certain  forms,  a  condition  which 
is  different  from  any  known  protozoan.  As  before  stated,  there 
is  so  much  discordance  in  the  published  work  of  the  various  in- 
vestigators that  definite  conclusions  cannot  be  reached.  It  has 
been  suggested  that  these  organisms  form  a  group  intermediate 
between  the  bacteria  and  the  protozoa.  This  is  not  as  probable  as 
has  been  sometimes  urged,  and  such  a  disposition  is  simply  a  con- 
fession of  our  lack  of  definite  knowledge.  It  is  by  no  means 
improbable  that  some  species  of  this  group  will  be  found  to  belong 
to  the  bacteria  and  others  to  the  protozoa,  and  that  supposed 
homologies  are  only  superficial  resemblances. 

Certain  species  have  been  removed  from  the  genus  Spirochaeta 
and  new  genera  created  for  them  by  certain  authors.  These 
names  are  so  common  in  literature  that  their  meaning  should  be 
known.  It  may  here  be  again  emphasized  that  the  name  Spiro- 
chceta, as  used  in  the  discussion  of  the  various  species  below, 
includes  all  of  these  genera. 

Spirochceta  (In  Narrow  Sense). — Organism  with  an  exceedingly 
slender,  spiral,  flattened  body,  with  a  narrow,  undulating  membrane 
that  surrounds  the  entire  body  in  a  spiral.  Xo  flagella  and  no 
spores  are  produced.  Reproduction  probably  occurs  by  longi- 
tudinal division. 

Spiroschaudinnia. — Blood  parasites.  Minute  wavy  or  spiral 
threads,  with  undulating  membrane  and  no  flagella.  Free  motile 
stage  alternates  with  a  stage  in  which  the  organism  is  coiled  up 
in  one  of  the  host-cells.  Developmental  stages  have  been  demon- 
strated in  the  intermediate  host.  The  life-history  is  imperfectly 
known. 

Treponema. — Spiral  body,  not  flattened,  tapering  at  the  ends. 
Flagellum  at  each  pole.  No  undulating  membrane.  Multiplica- 
tion by  longitudinal  division.  Does  not  stain  with  common 
aqueous  anilin  dyes:  special  staining  methods  are  required. 

The  following  organisms  described  as  belonging  to  this  group 
are  of  sufficient  economic  importance  to  warrant  their  con- 
sideration here:  Spirochceta  obermeieri,  Sp.  duttoni,  Sp.  pal- 
lida,  Sp.  pertenius,  in  man;  Sp.  anserina  (Sp.  gallinarwn),  in 


444  VETERINARY    BACTERIOLOGY 

geese  and  other  fowls;   Sp.  theileri,  in  cattle;  and  Sp.  ovina,  in 
sheep. 

All  the  members  of  this  group  may  be  characterized  as  slender, 
spiral  threads,  motile  by  sinuous  movements,  incapable  of  cultiva- 
tion on  ordinary  media,  and  in  some  cases  difficult  to  stain, 
requiring  special  technic. 

Spirochaeta  obermeieri 

Synonyms. — Spiroschaudinnia  recurrentis;  Spirillum  obermeieri; 
spirillum  recurrentis. 

Diseases  Produced. — Relapsing  fever,  recurrent  fever,  spirillosis 
in  man. 

Obermeier,  in  1873,  published  his  discovery  of  a  large  spiral 
organism  in  the  blood  of  patients  suffering  from  relapsing  fever. 
Since  that  time  it  has  been  repeatedly  observed,  and  several 
similar  species  have  been  described  infecting  man,  differing  prin- 
cipally in  their  pathogenicity  for  small  animals,  and  the  fact  that 
immunity  to  one  does  not  immunize  against  the  other. 

Distribution. — The  disease  is  known  from  Europe,  and  occurs 
in  isolated  cases  in  many  parts  of  the  world. 

Morphology  and  Staining. — Spirochceta  obermeieri  is  a  very 
slender,  tapering  spiral.  It  is  about  0.4  u  in  diameter,  and  varies 
greatly  in  length.  It  is  always  many  times  as  long  as  broad. 
There  are  from  two  to  ten  spirals  or  turns  in  the  organism,  as 
commonly  observed.  Opinions  differ  as  to  the  presence  of  flagclla 
and  undulating  membrane.  It  is  motile,  with  a  very  rapid,  screw- 
like  motion  and  a  waving  motion  of  the  entire  organism.  The 
organism  may  be  observed  in  the  living  condition.  It  is  best 
stained  by  the  Romanowsky  method  or  some  modification  of  it. 

Isolation  and  Culture. — The  organism  has  never  been  success- 
fully cultivated  upon  ordinary  media.  Some  multiplication  may 
take  place  in  freshly  drawn  blood,  but  it  does  not  long  continue, 
and  its  occurrence  has  been  denied  by  some  investigators. 

Pathogenesis. — Experimental  Evidence. — The  organism  is  path- 
ogenic for  man,  monkeys,  mice,  and  rats.  The  only  practicable 
method  of  maintaining  a  culture  is  by  repeated  transfers  of  the 
organism  from  one  animal  to  another. 

Character  of  Disease  and  'Lesions. — The  disease  in  man  is  char- 


SPIROCHETE   GROUP  445 

acterized  by  pains  in  the  head  and  back  and  by  high  fever.  This 
is  followed  by  a  complete  apparent  recovery.  A  second  attack,  or 
relapse,  occurs  usually  in  about  a  week,  then  a  third,  and  even 
more.  The  relapses  tend  to  decrease  in  intensity.  The  spiro- 
chetes  are  to  be  found  in  large  numbers  during  the  relapses,  though 
usually  in  diminished  numbers. 

Immunity. — No  specific  toxin  has  been  demonstrated.  An 
attack  of  the  disease  confers  an  active  immunity.  Repeated  in- 
jections of  blood  containing  spirochetes  result,  in  the  rat,  in  the 


v 


Fig.  196. — Spirochceta  ooermeieri  from  the  blood  of  a  rat  (Novy  and  Knapp 
in  "Journal  of  Infectious  Diseases").' 

development  of  a  hyperimmunity.  Immune  and  hyperimmune 
blood  may  be  used  in  conferring  a  passive  immunity.  The  agen- 
cies responsible  for  the  development  of  immunity  are  not  well 
understood. 

Bacteriological  Diagnosis. — The  organism  may  be  found  in 
fresh  preparation  or  in  stained  mounts  of  the  blood  during  a 
paroxysm. 

Transmission. — The  disease  is  supposed  to  be  transmitted  by 
the  bite  of  an  infected  bed-bug  (Acanthia  lectularia) ,  possibly  by 


446  VETERINARY    BACTERIOLOGY 

the  body  louse  (Pediculus  vestimenti).  Whether  or  not  there  is 
a  developmental  cycle  in  the  body  of  the  parasite  is  not  certainly 
known. 

Spirochaeta  duttoni 

Synonyms. — Spiroschaudinnia  duttoni;  Spirillum  duttoni. 

Disease  Produced. — West  African  tick  fever  in  man. 

Ross  and  Milne,  and  Button  and  Todd,  working  independently 
in  1904,  demonstrated  the  presence  of  this  spirochete  in  the  blood 
of  individuals  infected  with  this  disease.  Morphologically,  it 


Fig.  197. — Spirochata  duttoni  from  the  blood  of  a  rat  (Novy  and  Knapp,  in 
"Journal  of  Infectious  Diseases"). 

resembles  the  preceding  closely,  but  is  usually  longer  and  more 
loosely  coiled.  It  is  said  to  possess  diffuse  flagella.  The  disease 
is  transmitted  by  the  bite  of  a  tick  (Ornithodorus  moubata). 

Duval,  Todd,  McGell,  and  Pong  have  reported  success  in  the 
cultivation  of  the  organism  outside  the  body.  Many  media 
were  used  with  uniform  failure  until  an  egg-yolk  preparation  was 
found  to  be  successful.  The  following  is  the  medium  recommended 
by  these  investigators.  Six  mice  are  skinned  and  their  bodies 
boiled  in  500  c.c.  of  water.  This  is  filtered  and  sterilized.  Two 


SPIROCHETE   GROUP  447 

egg  yolks  are  removed  under  aseptic  precautions  to  a  sterile  Erlen- 
meyer  flask.  One  hundred  c.c.  of  the  sterile  mouse  decoction  is 
mixed  with  these  and  shaken  thoroughly;  5  c.c.  of  sterile  de- 
fibrinated  mouse  blood  is  then  added.  The  flask  is  sealed  to  pre- 
vent evaporation,  and  placed  at  37  °  for  six  or  eight  weeks,  during 
which  time  the  material  undergoes  autolytic  digestion.  If  properly 
prepared,  the  digestion  is  brought  about  wholly  by  autolytic 
enzymes,  and  is  not  due  to  the  presence  of  bacteria.  Examination 
of  the  flask  at  the  end  of  the  digestion  should  show  it  still  to  be 
sterile.  The  material  for  inoculation  is  secured  by  removing 
heart  blood  from  an  infected  mouse,  using  aseptic  precautions. 
They  found  that  the  organism  multiplied  and  retained  its  viru- 
lence for  forty  days  under  these  conditions. 

Life  Cycle.— The  work  of  Leishman,  Todd,  and  others  indi- 
cates that  the  spirochetes,  when  taken  into  the  body  of  the  tick, 
undergo  a  kind  of  nuclear  fragmentation  into  chromatin  granules, 
which  find  their  way  through  the  wall  of  the  digestive  tract  into 
the  various  ofgans,  principally  to  the  Malpighian  tubules  and  the 
ovaries.  These  same  granules  may  be  demonstrated  in  the  eggs 
of  infected  females,  and  the  nymphs  are  able  to  transmit  the  disease. 
Leishman  believes  that  holding  the  tick  at  a  temperature  of  34°, 
or  even  blood  heat,  causes  these  granules  to  develop  into  small 
spirochetes.  They  are  exuded  from  the  body  through  the  coxal 
glands  and  the  fluid  secretion  of  the  Malpighian  tubules,  and  gain 
entrance  to  the  wound  caused  by  the  tick  bite  after  the  tick  has 
loosed  its  hold,  and  not  through  the  salivary  glands.  It  is  pos- 
sible that  a  cycle  of  changes  of  this  type  may  account  for  the 
relapses  that  occur  in  the  disease. 

Immunity. — Leishman  succeeded  in  establishing  an  active 
immunity  in  a  monkey  that  recovered  from  the  disease  by  causing 
infected  ticks  to  feed  upon  it  at  intervals. 

Spirochaeta  kochi 

This  organism,  causing  East  African  tick  fever,  has  been  found 
to  be  distinct  from  that  causing  the  West  Coast  fever.  To  the 
former  type  the  name  Spirochceta  kochi  has  been  given.  Other 
related  types  of  organisms  'causing  relapsing  fever  have  been 
described;  that  described  by  Xovy  and  Knapp  has  been  called 


448  VETERINARY    BACTERIOLOGY 

Spirochceta  novyi.     It  is  probable  that  a  disease  found  in  India 
is  caused  by  still  another  organism. 

Spirochaeta  anscrina  (or  Gallinartmi) 

Synonyms. — Spirillum  anserina;  Spirochceta  gallinarum;  Sp. 
Marchouxi;  Sp.  nicottei. 

Disease  Produced. — Spirochetosis,  spirillosis,  or  septicemia  in 
^  geese  and  domestic  fowls. 

Sacharoff,  in  1890,  described  the  Spirochceta  anserina  as  the 
cause  of  an  acute  septicemia  in  geese,  and  the  same  organism  was 
studied  by  Gabritschewsky  in  1898.  Marchoux  and  Salimbeni, 
in  1903,  reported  a  septicemia  or  spirochetosis  of  domestic  fowls 
in  Brazil.  Since  that  time  similar  organisms  have  been  reported 
from  many  places.  There  is  considerable  difference  of  opinion 
as  to  the  identity  of  the  spirochetes  isolated  from  the  goose  and 
the  domestic  fowl,  and  from  the  latter  in  various  parts  of  the  world. 
The  peculiarities  in  virulence  are  such  that  the  problem  can  be 
solved  only  with  considerable  difficulty.  The  organisms  are 
morphologically  identical.  They  are  at  least  closely  related,  and 
are,  therefore,  grouped  together.  The  name  Sp.  anserina  was  the 
one  first  used,  and,  therefore,  has  priority.  The  name  Sp.  gal- 
linarum, however,  is  more  commonly  met  with  in  literature. 

Distribution. — The  disease  has  been  reported  from  Russia  and 
the  Caucasus,  northern  central,  and  southern  Africa,  southern 
Asia,  and  South  America.  It  probably  is  wide-spread. 

Morphology  and  Staining. — The  organism  of  fowl  spirillosis  is  a 
tenuous  spiral  10  to  20  u  in  length,  with  an  average  of  one  spiral 
per  micron  in  length.  It  is  actively  motile.  No  flagella  have 
been  demonstrated.  According  to  Balfour,  the  organism  under- 
goes changes  in  the  body  of  the  intermediary  host,  the  tick  (Argas 
persicus)  corresponding  closely  to  those  described  above  for  Sp. 
duttoni  in  the  tick  (Ornithodorus  moubata).  The  organism  pro- 
duces the  same  characteristic  chromatin  granules.  Chicks  in- 
oculated with  material  showing  these  granules,  but  no  spirochetes, 
are  found  to  develop  typical  spirochetosis. 

The  organism  may  be  easily  demonstrated  when  living -and 
motile.  Carbol-fuchsin,  Leishman,*  and  Giemsa's  stains  may  be 
in  the  preparation  of  mounts. 


SPIROCHETE    GROUP  449 

Isolation  and  Culture. — The  organism  has  not  been  cultivated 
upon  artificial  media. 

Pathogenesis. — Experimental  Evidence. — Many  birds  may  be 
infected  by  inoculation,  among  them  the  goose,  duck,  fowl,  guinea 
fowl,  turtle-dove,  sparrow,  and  other  birds;  usually  not  the  pigeon, 
although  there  is  disagreement  on  this  point.  The  rabbit,  white 
mouse,  guinea-pig,  monkey,  horse,  and  man  are  not  susceptible. 
Each  of  the  various  types  described  have  usually  showed  the 
greatest  virulence  for  the  species  of  bird  from  which  it  was  originally 
described,  and  many  variations  in  virulence  have  been  observed. 


f 


Fig.  198. — Spirochaeta  anserina  (gaUinarum) .     An  agglutinated   group  from 
the  blood  of  a  fowl  (Xovy  and  Knapp,  in  "  Journal  of  Infectious  Diseases  ")• 

It  is  believed  that  transfer  through  the  intermediary  host  is  neces- 
sary for  the  retention  of  virulence. 

Character  of  Disease  and  Lesions. — The  disease  is  characterized 
as  a  true  septicemia;  Levaditi  has  shown  that  the  organisms  also 
invade  the  intercellular  spaces  in  various  organs.  The  disease 
runs  an  acute  course,  marked  by  fever.  Young  fowls  may  show 
relapses,  but  the  adults  rarely.  In  this  respect  the  disease  differs 
from  that  caused  by  Spirochceta  obermeiei'i  in  man.  The  organisms 
are  present  in  the  blood  in  great  numbers  during  the  crisis.  They 
rapidly  disappear  during  convalescence.  The  mortality  varies 

29 


450  VETERINARY  BACTERIOLOGY 

from  80  per  cent,  or  more  in  some  outbreaks  to  a  small  percentage 
in  others. 

Immunity. — It  is  probable  that  agglutinins  are  formed,  but  the 
difficulty  of  getting  suspensions  of  the  spirochete  is  so  great  that 
the  question  has  not  been  adequately  tested.  Immunity  is 
developed  by  an  attack  of  the  disease  followed  by  recovery,  but 
to  what  agencies  this  is  due  is  not  certainly  known.  Marchoux 
and  Salimbeni  heated  blood  from  infected  fowls  for  five  minutes  at 
55°,  and  succeeded  in  establishing  immunity  by  its  injection. 

Transmission. — The  disease  is  commonly  transmitted  from  one 
fowl  to  another  by  the  ticks  Argas  persicus,  Argas  miniatus,  and 
possibly  Argas  reflexus.  The  louse  is  believed  also,  by  Balfour, 
to  be  an  intermediary  host.  The  latter  author  found  that  spiro- 
chetes  could  be  demonstrated  in  the  salivary  glands  of  the  tick  in 
fourteen  days  after  injection.  The  necessity  for  a  definite  incuba- 
tion period  has  not  been  shown. 

Spirochaeta  theileri 

Synonyms. — Spirillum  theileri;  Spirillum  ovina;  Spirochceta 
ovis;  Spirochceta  equi. 

Disease  Produced. — Spirillosis  in  cattle,  sheep,  and  horses. 

Theiler,  in  1902,  discovered  a  spirochete  in  the  blood  of  cattle 
in  South  Africa.  It  has  been  found  since  that  time  in  Cameroon, 
East  Africa,  and  Annam.  The  organism  is  0.25  to  0.4  p  by  10  to 
30  ^.  It  resembles  the  preceding  morphologically.  The  disease 
is  a  benign  infection,  and  the  organisms  soon  disappear.  It  is 
transmitted  by  the  bite  of  a  tick  (Rhipicephalus  decolor atus) . 
Theiler,  in  1904,  reported  the  occurrence  of  a  spirochete  similar 
to  Spirochceta  theileri  in  the  blood  of  sheep  in  the  Transvaal. 
It  was  later  found  in  northern  Africa.  Novy  and  Knapp  have  pro- 
posed the  name  Spirillum  (Spirochceta)  ovis  for  this  form.  The 
same  investigator  described  a  spirochete  associated  with  a  disease 
of  the  horse  in  South  Africa,  and  later  it  was  reported  from  the 
west  coast.  Novy  and  Knapp  term  this  Spirochceta  equi.  Todd 
and  others,  by  cross  inoculations,  have  demonstrated  that  these 
organisms  from  the  horse,  sheep,  and  ox  all  belong  to  the  same 
species. 


SPIROCHETE  GROUP  451 

Spirochxta  pallida 

Synonyms. — Spirillum  pallidum;  Treponema  pallidum. 

Disease  Produced. — Syphilis  in  man. 

Schaudinn  and  Hoffmann,  in  1905,  described  the  organism  which 
is  now  generally  accepted  as  the  cause  of  syphilis.  Prior  to  that 
date  many  organisms  had  been  described  as  possible  causes, 
but  all  are  now  known  either  to  be  secondary  invaders  or  commen- 
sals. 

Distribution. — The  disease  is  widely  distributed  among  all 
civilized  peoples. 

Morphology  and  Staining. — The  Spirochceta  pallida  is  a  slender 
organism,  less  than  0.5  ^  in  diameter  and  4  to  20  ^  in  length. 


f_i^      .   f  ^^_ 

tA      v 

JM  j 

^ 


r 


Fig.  199. — SpirochcBta  pallida  in  the  lumen  of  a  bronchus  in  congenital  syphilis 

(Hedren). 


The  spirals  are  quite  regular,  and  vary  in  number  from  three  to 
forty.  A  flagellum  has  been  detected  at  each  pole.  The  organism 
is  actively  motile.  Multiplication  is  probably  by  transverse 
division,  although  longitudinal  division  is  said  to  occur.  The 
organism  is  so  slender,  and  stains  with  such  difficulty,  that  there 


452  VETERINARY   BACTERIOLOGY 

are  many  unsettled  points  relative  to  its  morphology.  Whether 
or  not  it  may  pass  through  a  cycle  of  changes  that  would  mark  it 
as  certainly  protozoan  is  not  definitely  known. 

The  common  anilin  dyes  used  in  bacteriological  work  fail  to 
demonstrate  this  organism  in  tissues  or  in  smears.  The  India- 
ink  method  of  demonstrating  spirochetes  may  be  used  as  a  simple 
procedure  for  showing  the  organisms  in  smears.  This  consists  in 
allowing  a  thin  film  of  India  ink  to  dry  upon  the  smear.  The 
organisms  do  not  take  up  the  ink,  and  may  be  recognized  as 
transparent  spirals  in  the  black  field.  They  may  also  be  stained 
by  Giemsa's  eosin  and  azur  stains.  The  stain  must  be  freshly 
prepared.  Very  satisfactory  results  are  achieved  by  impregna- 
tion with  silver,  particularly  for  the  demonstration  of  the  organ- 
ism in  tissues.  t 

Isolation  and  Culture. — The  organism  has  never  been  cultivated 
upon  artificial  media. 

Pathogenesis. — Experimental  Evidence. — The  organism  may 
always  be  found  in  the  primary  and  secondary  lesions  of  syphilis, 
and  has  been  repeatedly  demonstrated  in  the  tertiary  as  well, 
although  it  is  much  more  difficult  to  find  in  the  latter  stage.  It 
may  be  found  in  the  internal  organs  of  a  syphilitic  fetus.  An 
infection  has  been  produced  in  the  cornea  and  the  iris  of  the  rabbit, 
and  the  organism  shown  to  be  present.  The  primary  and  secon- 
dary lesions  of  the  disease  have  been  produced  in  the  monkey, 
particularly  in  the  anthropoid  apes,  and  the  spirochetes  found  in 
each  of  the  stages.  The  evidence  is  very  strong,  therefore,  that 
Spirochaeta  pallida  is  the  cause  of  syphilis.  Until  the  organisms 
can  be  injected  in  pure  cultures  and  produce  the  disease  this  evi- 
dence cannot  become  indisputable,  however. 

Character  of  Disease  and  Lesions. — In  man  the  primary  lesion 
in  the  form  of  a  chancre  appears  in  about  three  weeks  after  in- 
fection, usually  on  or  near  the  external  genitalia.  It  is  followed  by 
invasion  of  the  neighboring  lymphatics  and  by  progressive  en- 
largement of  the  lymph-nodes  as  the  disease  progresses.  Usually 
about  six  weeks  elapse  between  the  appearance  of  the  primary 
and  secondary  lesions.  These  latter  are  probably  dependent 
upon  an  invasion  of  the  blood,  and  consist  of  localized  skin  erup- 
tions, falling  of  the  hair  (alopecia),  and  the  symptoms  of  generalized 


SPIROCHETE   GROUP  453 

infection,  such  as  fever.  This  may  last  for  several  years  and 
an  immunity  be  established,  which,  however,  may  not  be  complete 
enough  to  prevent  gradual  sclerosis  of  blood-vessel  walls  and  de- 
generations in  the  parenchymatous  organs,  and  even  the  appear- 
ance of  tertiary  lesions. 

Immunity. — No  practicable  method  of  either  active  or  passive 
immunization  against  the  disease  has  been  developed  by  the  use 
of  the  organism  or  its  products. 

Bacteriological  Diagnosis. — This  may  be  accomplished  by 
direct  examination,  stained  mounts,  the  Wassermann  test,  or 
by  chemical  recognition  of  certain  changes  in  the  character  and 
composition  of  the  blood-serum.  The  organisms  may  be  observed 
in  the  fluid  expressed  from  fresh  tissues  by  use  of  dark  field  illu- 
mination. Smears  may  be  prepared  and  stained  with  Giemsa's 
stain,  or  tissue  sections  may  be  used. 

The  Wassermann  test  for  syphilis  has  already  been  described 
in  the  discussion  of  fixation  of  complement  in  the  section  on  Im- 
munity. As  an  antigen,  extracts  from  the  organs  of  a  fetus  are 
used,  for  in  these  organs  the  spirochetes  are  found  in  the  greatest 
numbers.  The  blood-serum  of  the  suspected  patient  is  tested 
for  its  possible  content  of  specific  amboceptor  with  fresh  guinea- 
pig  serum  for  complement,  sheep  red  blood-cells,  and  the  serum 
from  a  rabbit  possessing  hemolytic  amboceptor  for  these  erythro- 
cytes.  The  test  requires  considerable  care  and  must  be  checked 
at  every  step.  It  has  been  found  in  practice  to  give  quite  reliable 
data.  The  test  has  been  modified  in  many  ways  since  first  pro- 
posed. 

Various  substances,  such  as  1  per  cent,  solutions  of  lecithin, 
sodium  oleate,  sodium  glycocholate,  and  taurine  have  been  found 
to  give  more  or  less  characteristic  precipitates  with  the  blood  of 
syphilitics. 

Transmission. — The  disease  is  transmitted  usually  through 
sexual  congress,  rarely  through  infective  drinking  vessels,  closets, 
and  by  direct  inoculation,  as  sometimes  happens  in  surgical  work. 
The  disease  may  be  inherited :  the  organism  may  possibly  enter 
through  the  ovum  or  the  sperm,  or  pass  from  the  circulation  of  the 
mother  to  that  of  the  fetus. 


454  VETERINARY   BACTERIOLOGY 

Spirochaeta  pertenois 

Synonym. — Treponema  pertenuis. 

Disease  Produced. — Yaws  in  man. 

Castellani,  in  1905,  reported  the  occurrence  of  spirochetes  in  a 
tropical  disease  known  as  yaws.  The  organism  resembles  that 
of  syphilis,  but  is  probably  distinct,  as  shown  by  inoculation 
experiments  and  study  of  specific  antigens  and  antibodies  in  com- 
parison with  those  of  syphilis. 

OTHER  SPIROCHETES 

Dodd,  in  1906,  found  a  spirochete  in  a  disease  of  the  pig  in 
South  Africa.  It  was  associated  principally  with  dark,  hemor- 
rhagic  lesions  of  the  skin. 

Spirochetes  may  be  found  in  considerable  numbers  in  the 
mouth,  and,  under  certain  conditions,  in  the  intestinal  tract,  upon 
the  skin,  and  about  the  genitalia.  They  are,  for  the  most  part, 
believed  to  be  harmless  commensals. 


CHAPTER  XLIII 

SPOROZOA 

THE  Sporozoa  stand  alone  among  the  protozoa,  in  that  all 
the  species  are  parasites.  Many  are  harmless  commensals,  but  a 
considerable  number  are  pathogenic.  The  sporozoan  cell  contains 
typically  a  single  nucleus,  except  in  the  Myxosporidia  which  are 
multinucleate.  Food  is  taken  in  by  diffusion  through  the  plasma 
wall.  Gastric  and  contractile  vacuoles  are  present.  The  adult 
form  is  non-motile;  the  young  forms  are  frequently  actively 
motile,  either  ameboid  or  flagellate.  In  most  of  the  Sporozoa 
the  differentiation  of  the  protoplasm  into  ectoplasm  and  endo- 
plasm  can  be  clearly  made  out  The  reproduction  of  the  Sporozoa 
is  the  principal  character  which  differentiates  this  group  from 
others.  Spores  are  always  produced  in  those  forms  in  which  the 
complete  life-history  is  known.  The  details  of  spore-formation 
vary  greatly  in  the  different  forms,  but  the  essentials  are  the  same. 

The  cell  as  a  whole,  in  most  forms,  divides  to  form  archispores 
or  sporoblasts ;  each  of  the  archispores  forms  spores,  and  each  spore 
then  produces  one  or  more  sporozoites.  Many  forms  show  addi- 
tional methods  of  reproduction.  Formation  of  sex  cells  or  gametes, 
with  fusion  of  like  or  unlike  cells,  takes  place  in  many  forms,  and 
serves  to  further  complicate  the  life-history. 

Blood-smears  may  be  stained  by  one  of  the  Romanowsky 
stains  or  by  Wright's  method  to  demonstrate  the  protozoa  of  this 
group.  Considerable  care  must  be  exercised  in  the  examination 
of  such  blood  mounts  not  to  confuse  the  normal  blood  contents, 
such  as  blood-platelets,  with  developmental  stages  of  the  protozoa. 

The  class  Sporozoa  contains  many  families  and  genera;  only 
a  few  of  the  latter  contain  species  that  are  of  economic  importance. 
The  following  artificial  key  will  assist  in  differentiating  the  various 
genera. 

455 


456  VETERINARY   BACTERIOLOGY 

A.  Sporozoa  found  in  the  blood -cells. 

1.  In  the  erythrocytes. 

a.  At  some  stage  occupying  a  considerable  proportion  of  the  interior  of 

the  cell. 

(1)  In  mammalian  blood. 

Well-differentiated,  usually  pear-shaped  bodies,  often  two  in  a  cell. 

In   animals Piroplasma  or  Babesia. 

Ameboid  at  first,  finally  filling  the  cell.     In  man Plasmodium. 

(2)  In  blood  of  birds Proteosoma,  Halteridium,  Hemoproteus, 

b.  Forming  minute  dots,  seemingly  entirely  of  chromatin.  .  .Anaplasma. 

2.  In  the  leukocytes Leukocytozoon. 

B.  Sporozoa  in  muscles Sarcocystitis  and  Balbiana. 

C.  Sporozoa  usually  hi  membranes  (mucous  or  serous) Coccidium. 

THE  GENUS  PIROPLASMA,  OR  BABESIA 

An  organism  belonging  to  this  genus  was  first  noted  by  Babes 
and  called  by  him  Haematococcus.  Theobald  Smith,  in  1889,  made 
the  first  observation  which  related  one  of  these  organisms  to 
Texas  fever.  He  called  the  organism  Pyrosoma.  This  was  later 
changed  to  Piroplasma  by  Patton,  and  still  later  by  Starcovici  to 
Babesia. 

The  organisms  of  this  genus  occur  in  the  red  blood-cells  of 
various  mammals  and  produce  several  distinct  diseases.  The  life- 
history  of  all  the  species  has  not  been  satisfactorily  worked  out. 

Piroplasma  bigeminum 

Synonyms. — Pyrosoma  bigeminum;  Apiosoma  bigeminum;  Babe- 
sia bigeminum  bovis;  Hcematococcus  bovis;  Ixidoplasma  bigeminum. 

Disease  Produced. — Texas  fever,  or  tick  fever  in  cattle — bovine 
piroplasmosis. 

Theobald  Smith,  in  1889,  discovered  the  cause  of  Texas  fever 
in  cattle.  His  work  was  fundamental  and  remarkably  complete1. 
Since  that  time  investigators  have  found  the  same  organims  in 
many  countries. 

Distribution. — Southern  United  States,  Australia,  Argentina, 
Europe,  India,  and  Africa. 

Morphology. — In  the  blood  of  infected  animals  the  organisms 
are  generally  in  pairs.  They  are  commonlv  piriform,  one  end  In  MI  IK 
rounded  and  the  other  somewhat  pointed.  The  acute  ends  are 
usually  pointed  toward  each  other.  The  organisms  vary  from 
0.5  to  4  (A  in  diameter.  The  reproductive  stages  have  not  been 


SPOROZOA  457 

thoroughly  worked  out.  The  organism  stains  readily  with  such 
dyes  as  alkaline  methylene-blue  and  by  Wright's  method. 

Pathogenesis. — The  relationship  of  the  organism  to  the  disease 
has  been  satisfactorily  demonstrated  by  inoculation  experiments. 
The  disease  in  cattle  is  characterized  by  fever  and  a  hemoglobin- 
uria,  with  considerable  destruction  of  red  blood-corpuscles.  In 
acute  cases  death  often  occurs  in  five  to  eight  days  after  the 
symptoms  are  first  noted.  Those  cases  in  which  recovery  takes 
place  generally  harbor  still  in  their  bodies  the  specific  organisms, 
but  remain  perfectly  well. 

Immunity. — As  already  noted,  recovery  from  disease  does  not 
necessarily  predicate  the  disappearance  of  the  organism  from  the 


Fig.  200. — Piroplasma  bigeminum,  infected  red  blood  cells  with  1-4  parasites, 
a  blood-platelet  in  the  center  (Sieber). 

blood,  and  it  may  persist  for  years.  It  has  been  found  that 
immunity  against  a  fatal  attack  of  this  disease  may  be  conferred 
by  inoculation  with  the  blood  of  immune  animals.  This  results 
generally  in  a  mild  infection,  which  immunizes  against  one  of  a 
severer  type.  Such  methods  of  vaccination  are  quite  widely 
practised. 

Bacteriological  Diagnosis. — The  organism  may  usually  be 
demonstrated  in  the  blood  when  stained  with  Loffler's  methylene- 
blue  or  Wright's  stain. 

Transmission. — Natural  infection  takes  place  only  through  the 
bite  of  infected  cattle  ticks  (Rhipicephalus  annulatus  or  Boophilus 
bovis)  in  the  United  States  and  closely  related  forms  in  other 


458  VETERINARY   BACTERIOLOGY 

countries.  The  female  tick  becomes  engorged  with  blood  and  falls 
to  the  ground,  where,  after  a  time,  eggs  are  laid.  They  hatch  in 
from  nineteen  days  to  five  or  six  months,  depending  upon  the 
temperature  conditions.  The  young  ticks  crawl  up  the  stems  of 
grass  and  shrubs.  They  must  get  upon  the  body  of  an  animal  or 
die  of  starvation.  The  ticks  from  an  infected  mother  are  them- 
selves infective,  and  may  transmit  the  disease  to  the  animal  whose 
blood  they  suck. 

Piroplasma  parvum 

Synonyms. — Babesia  parva;  Theileria  parva. 

Disease  Produced. — East  African  coast  fever,  Rhodesian  red- 
water,  Rhodesian  tick  fever  in  cattle. 

The  organism  and  the  disease  have  been  studied  by  Theiler, 
Koch,  and  others.  This  protozoan  is  the  smallest  of  the  Piro- 
plasmas  known.  In  the  red  cells  it  forms  a  small  rod  that  has 
a  chromatin  granule  at  one  end.  Frequently  ring  forms  are  ob- 
served, never  the  pear-shaped  types  of  P.  bigeminum.  Gonder 
has  worked  out  in  detail  the  life-history  of  the  organism.  This 
disease  is  peculiar,  in  that  transference  of  blood  containing  the  or- 
ganism from  one  animal  to  another  does  not  resu't  in  the  transfer- 
ence of  the  disease.  Repeated  inoculations  are  without  effect. 
It  is  transmitted  by  means  of  the  brown  t'ck  (Rhipicephalus  ap- 
pendiculatus)  and  the  black  pitted  tick  (Rh.  simus).  The  affected 
animal  shows  high  fever  and  swelling  of  the  lymph-nodes.  Anemia, 
icterus,  and  hemoglobinuria  are  rare'y  observed.  Immunity  to 
this  disease  does  not  immunize  against  Texas  fever.  What  is 
probably  the  same  disease  has  also  been  described  from  southern 
Russia  and  in  Java. 

Piroplasma  mutatis 

Theiler  has  established  the  presence  of  a  third  piroplasmosis 
in  southern  Africa,  due  to  a  protozoan  which  he  has  named  P. 
mutans.  It  is  smaller  than  the  P.  bigeminum,  and  animals  im- 
munized against  one  will  contract  the  other. 

Pfroplasma'  cqui 

Disease  Produced. — Equine  biliary  fever.  Equine  piroplas- 
mosis. 

Guglienni,   in   1899,   discovered  this  organism  in   Italy,  and 


SPOROZOA 


459 


r-    , 


«4T*j? 

•V  V. 


Fig.  201. — Piroplasma  parvum,  life  cycle:  1,  Agametes  of  the  first  generation 
(inetagametes) ;  2,  a  and  6,  agamonts  with  one  nucleus;  3,  a  and  6,  agamonta 
with  several  nuclei;  4,  a  and  6,  medium-sized  agamonts;  5,  a  and  b,  large  aga- 
monts with  numerous  nuclei;  6,  a  and  6,  agamonts  undergoing  schizogony;  7, 
a  and  6,  agametes;  8,  a  and  6,  reduction  forms  of  agamonts;  9,  a  and  6,  seg- 
mentation of  reduction  forms  of  agamonts;  10,  a  and  6,  young  agamonts;  11, 
a  and  6,  medium-sized  gamonts  with  several  nuclei;  12  and  13,  a  and  6,  large 
gamonts  with  numerous  nuclei;  14,  a  and  6,  gamonts  undergoing  schizogony; 
15,  free  gametocytes;  16,  gametocytes  in  the  red  blood-corpuscles;  17,  micro- 
and  macrogametes  in  the  stomach  of  the  tick;  18,  copulation;  19,  karyomyxis; 
20  and  21,  formation  of  the  ookinetes;  21,  retort  forms  of  ookinete;  22, 
ookinete  (Gonder,  in  "  Journal  of  Comparative  Pathology  and  Therapeutics"). 


460  VETERINARY   BACTERIOLOGY 

Theiler  later  elaborated  an  account  of  the  disease  and  the  organism 
as  it  occurs  in  South  Africa. 

Distribution. — The  disease  has  been  noted  from  South  Africa, 
Central  Africa,  Algeria,  Italy,  Sweden,  Russia,  India,  and  Vene- 
zuela. It  is  evident  that  further  observation  may  show  an  ex- 
tensive distribution 

Morphology. — The  organism  is  smaller  than  P.  bigeminum,  but 
resembles  it.  It  occurs  singly  or  in  pairs,  or  rarely  in  rosettes,  in 
the  red  blood-cells.  Occasionally  it  is  free  in  the  plasm.  The  disease 
was  at  first  supposed  to  be  non-transmissible  by  blood  injection, 
but  Theiler  succeeded  by  intravenous  injections  of  virulent  blood. 

Pathogenesis. — The  disease  is  characterized  by  jaundice  and  a 
high  fever.  It  may  run  an  acute  or  a  chronic  course;  frequently 
it  is  fatal  within  a  few  days.  The  lymph-nodes  and  the  spleen 
are  considerably  enlarged.  Animals  born  in  infected  districts 
are  commonly  immune. 

Transmission. — The  disease  is  believed  to  be  transmitted  by 
ticks. 

Piroplasma  ovis 

Disease  Produced. — Hemoglobinuria,  malarial  catarrhal  fever, 
or  icterohematuria  in  sheep. 

Babes,  in  1892,  first  noted  the  parasites  in  the  blood-cells  of 
sheep  in  Rumania  at  the  time  that  he  made  his  observations  on 
piroplasmosis  of  cattle. 

Distribution. — It  has  been  noted  from  Italy,  France,  Turkey, 
Venezuela,  and  the  West  Indies,  South  Africa,  Rumania,  and 
probably  in  the  United  States  (Montana). 

Morphology. — It  is  similar  to  P.  bigeminum,  but  smaller  (1  to 
1.8  ^  in  diameter).  It  is  commonly  single,  sometimes  double,  in  the 
cells,  and  frequently  occurs  in  the  plasm. 

Pathogenesis. — The  disease  may  be  transferred  by  the  injec- 
tion of  blood  containing  the  organisms  into  healthy  animals.  The 
incubation  period  is  about  eight  to  ten  days.  Other  animals, 
including  cattle,  cannot  be  infected.  The  disease  is  commonly 
fatal.  It  is  characterized  by  anemia,  icterus,  and  frequently 
hemoglobi  nuria. 

Transmission. — The  disease  is  transmitted  through  the  bite  of 
a  tick  (Rhipicephalus  bursa). 


SPOROZOA  461 

Piroplasma  canis 

Disease  Produced. — Biliary  fever  or  malignant  jaundice  of  the 
dog. 

Pram  and  Galli-Valerio,  in  1895,  first  described  the  blood 
parasite  of  canine  piroplasmosis. 

Distribution. — It  has  been  reported  in  China,  Italy,  France, 
Hungary,  South  Africa,. East  Africa,  and  possibly  from  the  United 
States. 

Morphology. — The  organisms  are  morphologically  almost 
identical  with  P.  bigeminum.  They  are  generally  2  to  4  n  in  diam- 
eter. They  are  sometimes  found  abundantly  in  the  plasma  and 
in  a  single  cell  there  may  be  as  many  as  sixteen  of  the  organ- 
isms. The  free  organisms  are  spherical;  those  within  the  cor- 
puscles are  pear-shaped  or  many  angled.  Multiplication  is  appar- 
ently by  direct  division. 


Fig.  202. — Piroplasma  canis:  1-11,  Organisms  in  various  developmental 
stages  in  red  blood-cells  in  culture;  12,  13,  organisms  free  in  the  plasm 
(Deseler). 

Pathogenesis. — The  disease  may  be  readily  transferred  by  the 
injection  of  virulent  blood.  It  cannot  be  transmitted  to  other 
species  of  animals.  Nuttall  and  Graham  Smith  did  not  succeed 
in  reproducing  the  disease  in  the  fox  and  jackal.  The  period  of 
incubation  is  three  days  or  more.  There  is  fever,  and  sometimes 
icterus  and  hemoglobinuria.  Anemia  is  marked.  The  spleen  is 
greatly  enlarged,  the  gall-bladder  is  distended,  and  the  kidneys 
are  often  ecchymotic.  Chronic  cases  frequently  recover.  The 
acute  cases  are  almost  invariably  fatal.  Animals  which  have 
apparently  recovered  retain  the  parasite  in  the  blood  for  long 
periods  and  retain  their  infect ivity. 

Bacteriological  Diagnosis. — Stained  blood-films  will  demon- 
strate the  organism  if  present. 


462  VETERINARY   BACTERIOLOGY 

Transmission. — At  least  three  species  of  tick,  and  probably  one 
species  of  flea,  have  been  found  to  act  as  carriers  of  the  organism. 

Piroplasma  gibsoni 

Patton  has  described  an  organism  causing  piroplasmosis  in 
hounds  in  the  Madras  Hunt  in  India.  Later,  it  was  discovered 
also  in  the  blood  of  a  native  jackal.  Its  relationship  to  the  P. 
canis  has  not  been  satisfactorily  determined. 

Piroplasma  commune 

Phillips  and  McCampbell  have  described  a  new  species  of 
Piroplasma  as  the  cause  of  an  epizootic  of  dogs  at  Columbus, 
Ohio.  The  organisms  found  were  similar  to  the  P.  cam's,  but  these 
investigators  were  able  to  demonstrate  the  organism  in  the  blood 
of  guinea-pigs  injected  with  virulent  blood.  The  cat  was  also 
infected,  but  not  the  horse,  cow,  rat,  or  rabbit.  This  fact  of 


Fig.  203. — Piroplasma  commune,  organisms  in  the  red  blood-cells  (adapted 
from  Phillips  and  McCampbell). 

transmissibility  led  to  the  tentative  adoption  of  a  new  specific 
name,  as  the  true  P.  canis  is  not  known  to  be  transmissible  to 
any  other  animals.  The  organisms  found  were  round  or  pear- 
shaped.  The  round  type  were  from  0.5  to  1.5  u  in  diameter,  and 
the  piriform  1.5  by  2.3  ^.  Considerable  pleomorphism  was 
evident. 

THE  GENUS  PLASMODIUM 

Malaria  in  man  has  been  found  to  be  due  to  three  species  of 
protozoa,  usually  placed  in  the  genus  Plasmodium.  The  organ- 
isms pass  certain  parts  of  their  life-cycle  in  the  blood-corpuscles 
in  man,  and  the  remainder  in  the  gut  and  tissues  of  the  mosquito. 
The  organisms  of  malaria  were  first  noted  by  Laveran  in  1880, 
and  the  various  types  have  been  differentiated  since  that  time. 


SPOROZOA 

Plasmodium  vivax 


463 


Disease  Produced. — Tertian  malaria  in  man. 
Distribution. — This   is   the   commoner   malaria   of   temperate 
climates. 


EXOGENOUS 

OR 

-3EXLJAU 


Fig.  204. — Diagram  illustrating  the  life-cycle  of  the  malarial  parasite: 
A,  Sporozoites  entering  a  red  blood-cell;  B,  C,  D,  E,  the  organism  in  various 
stages  of  development;  F,  G,  the  formation  of  sporocytes  and  their  division 
into  spores  which  infect  new  red  blood-cells.  The  series  A  to  H  represents 
the  cycle  through  which  the  organism  passes  in  the  human  body;  /,  infected 
red  cell  ingested  by  a  mosquito.  The  organism  may  now  develop  through  the 
series  «/',  K',  L',  M',  to  form  microgametocytes  and  microgametes  or  through 
/,  K,  L,  Af ,  to  form  a  macrogamete  which  unites  with  a  microgamete  to  form 
a  fertilized  ovum;  P,  the  organism  penetrates  the  stomach-wall  of  the  mos- 
quito and  develops  through  the  stages  Q,  R,  S,  T,  U.  From  U  large  numbers 
of  slender  spores  are  liberated  into  the  body  cavity.  These  pass  to  the  salivary 
glands  of  the  mosquito  and  are  injected  when  the  insect  again  bites  (Rees). 


Morphology  and  Life-history. — The  organism  when  first 
recognized  in  the  blood  is  small,  with  ameboid  movements.  It 
penetrates  the  red  blood-corpuscle,  and  develops  until  the  interior 


464  VETERINARY   BACTERIOLOGY 

of  the  corpuscle  is  filled.  When  full  grown,  it  may  double  the 
diameter  of  a  blood-cell.  The  organism  then  segments  to  form  a 
rosette  of  bodies,  which  round  off  to  form  small  spores  or  merozoites. 
These  are  freed  by  the  disintegration  of  the  red  blood-cell,  and 
attach  themselves  to  other  cells  and  begin  development  anew.  This 
may  be  repeated  several  times.  This  is  called  the  asexual  phase 
of  the  life-history.  The  organism  may  be  taken  in  by  the  mos- 
quito (Anopheles),  and  here  completes  its  life-cycle  by  passing 
through  the  sexual  phase.  Two  types  of  cells  are  found  to  de- 
velop from  the  spores  in  the  body  of  the  mosquito.  The  male 
cell  (known  as  microgametocyte)  produces  five  to  eight  micro- 
gametes.  The  female  cells  (macrogametes)  are  larger  and  granu- 
Jar.  A  microgamete  fuses  with  one  of  the  macrogametes  to  form 
what  may  be  termed  a  fertilized  "  egg,"  copula,  or  ookinete. 
This  burrows  into  the  wall  of  the  stomach  of  the  mosquito, 
encysts,  and  enlarges  greatly.  The  contents  finally  break  up 
into  a  considerable  number  of  spherical  bodies  known  as  sporo- 
blasts.  These  in  turn  produce  great  numbers  of  delicate  fila- 
mentous bodies  called  sporozoites.  These  are  liberated  by  the 
rupture  of  the  cyst,  and  pass  through  the  body  cavity,  and 
finally  enter  the  poison  or  salivary  gland,  whence  they  are 
inoculated  into  the  next  victim  of  the  mosquito.  This  cycle  in 
the  insect  is  completed  in  from  eight  to  ten  days,  and  during  this 
time  the  insect  is  not  infective. 

Pathogenesis. — The  disease  is  characterized  by  chills,  followed 
by  fever,  which  occur  every  forty-eight  hours.  The  infection  is 
usually  benign;  a  fatal  termination  is  very  rare.  The  chills  and 
fever  develop  at  the  time  of  formation  of  the  merozoiites  and  the 
infection  of  new  cells. 

Transmission. — The  disease  is  transmissible  only  through  the 
bite  of  the  mosquito.  The  elimination  of  the  possibility  of  this 
transfer  is  the  all-important  factor  in  efficient  prophylaxis. 

Plasmodium  malarias 

This  organism  produces  the  quartan  malaria  in  which  the 
interval  between  paroxysms  of  fever  is  seventy-two  hours,  and  tin- 
asexual  cycle  is  completed  in  this  time.  This  disease,  like  the 
preceding,  is  benign,  and  yields  readily  to  quinin  treatment. 


SPOROZOA  465 

Plasmodium  immaculatum  and  falciparum 

This  type  of  malaria  is  usually  tropical.  It  is  malignant, 
and  does  not  yield  readily  to  treatment.  Two  types  are  known, 
a  quotidian,  which  completes  its  asexual  cycle  in  twenty-four 
hours,  and  a  tertian,  which  requires  forty-eight.  Whether  or  not 
these  are  distinct  species  is  uncertain,  but  is  probable. 

THE  GENERA  PROTEOSOMA,  HALTERIDIUM,  AND  HEMOPROTEUS 

These  are  genera  of  sporozoa  which  produce  a  malaria-like 
infection  in  birds.  In  some  forms  a  part  of  the  life-cycle  is  also 
spent  in  the  body  of  the  mosquito — in  this  case  a  Culex.  None  of 
the  species  are  of  any  considerable  economic  importance. 

THE  GENUS  ANAPLASMA 

This  genus  was  created  by  Theiler  in  1910  to  accommodate  the 
organism  described  as  "  marginal  points  "  in  the  erythrocytes 
of  cattle.  The  protozoan  consists  of  a  tiny  dot  of  chromatin- 
like  material  in  the  corpuscle,  usually  near  the  margin,  never 
more  than  one-thirtieth  to  one-twentieth  the  size  of  the  cell. 
The  name  Anaplasma  comes  from  the  apparent  lack  of  any  cyto- 
plasmic  material. 

Anaplasma   marginale 

Synonym. — Marginal  points. 

Disease  Produced. — Anaplasmosis,  Galziekte,  or  gall  sickness 
in  cattle. 

This  organism,  according  to  Theiler,  has  been  observed  by 
several  investigators,  among  them  Smith  and  Kilborne,  in  their 
study  of  Texas  fever.  These  observers  have  believed  it  to  be  a 
developmental  stage  of  the  Piroplasma  (Babesia)  bigeminum. 
Theiler  has  succeeded  in  demonstrating  the  distinction  between 
the  organisms,  and  defines  Texas  fever  as  a  mixed  infection  of 
Anaplasma  marginale  and  Piroplasma  bigeminum. 

Distribution. — Known  with  certainty  from  South  Africa,  prob- 
ably widely  distributed. 

Morphology  and  Staining. — The  organism  may  be  single  in  the 
corpuscles,  or  there  may  be  several  within  a  single  cell.  The  para- 
sites usually  lie  near  the  periphery  of  the  corpuscle,  rarely  free 

30 


466  VETERINARY   BACTERIOLOGY 

in  the  blood.     They  are  small,  spherical,  rarely  more  than  one- 
tenth  of  the  diameter  of  the  cell,  frequently  less.     By  appropriate 


• .. 


Fig.  205. — Anaplasma  marginale  in  red  blood-cells.     Note  the  irregularity  in 
size  and  in  the  staining  of  these  cells  (Sieber). 

staining  methods  the  presence  of  a  central  granule  surrounded  by 
a  less  apparent  capsule  may  be  demonstrated.     Sieber  has  ob- 


Fig.  206. — Anaplasma  marginale,  stained  by  Heidenhain's  hematoxylon : 
a,  Single  parasite;  6,  beginning  of  division,  the  diplococcus  types;  c,  dumb-bell 
forms;  d,  completed  divisions;  e,  free  parasites  (Sieber). 

served  multiplication  of  the  organisms  within  the  blood-cells  by 
a  simple  type  of  division. 


SPOROZOA  467 

The  Anaplasma  marginale  does  not  stain  readily  with  the 
usual  anilin  dyes,  but  can  be  demonstrated  easily  by  a  stain 
such  as  Giemsa's.  Heidenhain's  iron  hematoxylon  also  gives 
good  results.  It  may  be  seen  even  in  the  living  cells  as  a  refrac- 
tive marginal  granule. 

Pathogenesis. — The  disease  produced  has  an  incubation  period 
of  sixteen  to  sixty  days.  The  specific  organisms  may  be  first 
demonstrated  from  the  spleen;  later  they  become  abundant  in  the 
blood.  As  many  as  30  per  cent,  of  the  cells  may  be  infected.  The 
first  evident  reaction  is  irregularity  and  poikilocytosis  of  the  red 
blood-cells,  followed  by  more  or  less  polychromasia  and  fragmenta- 
tion. The  serum  does  not  seem  to  acquire  any  hemolytic  proper- 
ties. The  febrile  periods  in  the  disease  coincide  with  the  presence 
of  the  greatest  number  of  marginal  points  in  the  corpuscles.  Cattle 
are  susceptible  to  artificial  infection.  It  is  believed  by  Theiler 
and  his  coworkers  that  the  primary  symptoms  of  disease  in  Texas 
fever  are  due  to  Piroplasma  bigemina,  but  that  the  secondary 
symptoms  are  due  to  this  Anaplasma  marginale,  which  requires 
a  longer  incubation  period. 

Transmission. — The  organism  is  transmitted  through  the  tick 
(Boophilus  decolor  atus) . 

THE. GENUS  LEUCOCYTOZOON 

Protozoa  somewhat  resembling  the  malarial  parasites  have 
been  found  in  the  white  blood-corpuscles  or  leukocytes  in  birds 


Fig.  207. — Hepatozoon  perniciosum,  a  leucocytozoon  from  the  blood  of 
a  rat:  a,  Free  parasite;  6,  parasites  in  the  mononuclear  leukocytes  (adapted 
from  Miller). 

by  several  observers,  and  in  the  domestic  fowl  and  the  dog  in 
Tonkin  by  Mathis  and  Leger.  They  are  not  known  to  be  of  any 
economic  importance. 


468 


VETERINARY   BACTERIOLOGY 


THE  GENUS  SARCOCYSTIS 

These  sporozoa  are  usually  elongated,  tubular,  oval,  or  even 
spherical.  Cysts  with  a  double  membrane  are  formed,  and  in  these 
are  produced  reniform  or  sickle-shaped  sporozo'ites,  with  a  polar 
capsule  and  a  projectile  thread. 

Species  of  this  genus  have  been  described  from  the  muscles 
of  a  large  number  of  vertebrates.  In  most  cases  the  organism 
does  not  do  any  appreciable  harm.  Recently  (1908)  Watson  has 
called  attention  to  the  prevalence  of  sarcosporidiosis  in  western 


Fig.  208. — Sarcocystis  muris  in  the  muscle  of  a  mouse:  1,  Mature  organism 
in  the  muscle;  2,  isolated  spores;  3,  4,  5,  stages  in  the  development  of  spores 
(adapted  from  Negri). 

Canada,  particularly  in  animals  suspected  of  loco  poisoning  or 
infected  with  dourine.  Six  cases  were  found  in  cattle  and  two  in 
horses  suspected  of  being  locoed,  three  in  dourine-affected  equines, 
and  one  in  a  filly  showing  cachexia.  He  concludes  that  these  or- 
ganisms may  sometimes  be  an  important  factor  in  disease.  In 
some  cases  the  entire  musculature  may  be  affected  with  serious 
and  even  fatal  consequences.  A  close  relationship  between  loco 
poisoning  and  sarcosporidiosis  is  shown  by  the  autopsy  records. 
Recovery  from  this  affection  and  from  dourine  may  be  prevented 
or  retarded  by  the  presence  of  the  organisms.  The  species  found 


SPOROZOA  469 

in  the  horse  is  Sarcocystis  bertrami;  in  sheep  and  goats,  S.  tenella; 
in  swine,  S.  miescheriana ;  and  in  man,  S.  lendemanni. 

THE  GENUS  COCCIDIUM 

This  genus  belongs  to  the  sporozoan  order  Coccidiida.  This 
order  is  characterized  by  having  in  the  adult  an  oval  or  spherical 
form,  not  motile.  Sporulation  takes  place  within  an  endocellular 
cyst.  The  genus  Coccidium  is  differentiated  by  the  formation 
of  four  sporocysts  or  sporoblasts,  each  of  which  contains  two 
sporozo'ites.  The  life-history  is  relatively  complex,  and  varies 
in  some  details  in  different  species. 

The  organism  is  taken  into  the  body  with  food  in  the  form 
of  a  cyst,  which  ruptures  and  allows  the  escape  of  the  spindle- 
shaped  sporozoites.  These  penetrate  the  epithelial  cells  of  the 
intestinal  walls  or  other  membranes.  The  sporozoTte  on  entering 
the  cell  rounds  up  into  a  sphere,  and  then  grows  rapidly  in  size 
at  the  expense  of  the  host  cell.  These  growing  organisms  are 
called  at  this  stage  schizonts.  The  nucleus  of  the  mature  schizont 
fragments,  and  the  protoplasm  then  breaks  up  into  a  considerable 
number  of  spindle-shaped  cells,  called  merozoites,  somewhat  re- 
sembling the  sporozoites.  These  break  out  of  the  mother  schizont 
and  infect  new  cells.  They  may  then  develop  as  schizonts  and 
repeat  the  same  cycle,  or  may  develop  into  sexual  reproductive 
cells.  Some  of  these  are  of  considerable  size  and  correspond  to 
an  egg;  these  are  termed  the  macrogametes.  Others,  called  micro- 
gametocytes,  develop  similarly  at  first,  then  form  a  considerable 
number  of  very  slender,  thread-like  cells  called  microgametes.  A 
microgamete  fuses  with  a  macrogamete  to  form  the  oocyte.  This 
then  continues  to  enlarge,  and  secretes  a  chitinous  wall;  i.  e., 
becomes  encysted.  When  mature,  the  contents  of  the  cyst  divide 
to  form  four  spherical  bodies  called  sporoblasts.  These  become 
somewhat  elongated  and  spindle-shaped.  In  each  of  the  sporo- 
blasts two  still  more  slender  fusiform  sporozoites  develop.  The 
cyst  is  freed,  and  upon  ingestion  by  a  suitable  host  the  cycle  be- 
gins again. 

Coccidiosis  occurs  in  many  of  the  invertebrates,  which  do  not 
seem  to  be  seriously  affected,  but  in  the  vertebrates  serious  and 
even  fatal  disease  may  be  caused  by  the  organisms. 


470  VETERINARY   BACTERIOLOGY 

Coccidium  tenellum 

Synonyms. — It  is  possible  that  this  organism  does  not  differ 
from  that  of  the  rabbit,  in  which  cases  the  name  given  would  be 
reduced  to  a  synonym  of  Coccidium  cuniculi. 

Disease  Produced. — Coccidiosis  in  domestic  fowls  and  in  birds, 
blackhead  or  enterohepatitis  in  turkeys,  white  diarrhea  (at  least 
some  types)  in  chicks,  possibly  roup  in  fowls. 

These  diseases  have  been  studied  by  a  great  number  of  inves- 
tigators, and  many  theories  of  their  causation  have  been  de- 
veloped. Hadley  and  others  have  seemed  recently  to  show  quite 
conclusively  that  they  are  cases  of  avian  coccidiosis,  and  the  various 
organisms  described  by  others  as  causal  were  secondary  invaders 
or  developmental  stages  of  the  organism  in  question. 

Distribution. — The  disease  probably  has  a  very  wide  distribu- 
tion over  the  United  States  and  Europe,  but  adequate  data  are 
not  at  hand  for  a  determination.  It  is  certainly  known  from 
many  localities  in  the  eastern  States. 

Morphology  and  Life-history. — The  life-history  is  typical  for 
Coccidium  as  outlined  above.  The  adult  coccidial  cyst  is  oval 
or  ellipsoidal.  It  measures  about  14  by  21  f*. 

Pathogenesis. — A  considerable  number  of  infections  in  the 
domestic  fowls  have  been  ascribed  to  this  organism.  A  mild  in- 
fection may  not  result  in  marked  symptoms.  An  intestinal  and 
cecal  infection  in  the  young  chick  has  been  found  to  be  a  potent, 
if  not  the  principal,  cause  of  white  diarrhea,  which  causes  such 
heavy  losses  in  certain  localities.  A  similar  infection  in  adult 
chickens  may  also  prove  fatal.  This  is  particularly  true  of  the 
turkey,  which  is  unusually  susceptible.  The  principal  symptoms 
are  three  in  number — diarrhea,  progressive  languor  or  stupor,  and 
loss  of  appetite  with  pronounced  emaciation.  The  disease  may 
be  acute  or  chronic,  but  is  quite  generally  fatal.  Some  fowls 
may  harbor  the  organisms  for  long  periods  without  any  apparent 
symptoms. 

Hadley  has  also  shown  that  this  same  organism  is  probably 
the  cause  of  roup.  In  this  disease  the  tissues  infected  are  the 
mucous  membranes  of  the  head.  The  infection  results  in  the 
"  inflammation  of  and  exudate  from  the  orbital  sinus,  nasal 
lachrymal  duct,  nasal  chamber,  mouth,  pharynx,  larynx  (some- 


SPOROZOA 


471 


times)  esophagus,  intestines,  and  ceca;  and  terminating  fatally 
in  the  majority  of  cases  as  a  result  of  a  series  of  combined  factors, 

I 

Z 


13 


IE 


Fig.  209. — Coccidium  tenellum,  life-cycle:  1,  Mature  encysted  Coccidium; 
2,  division  into  four  sporoblasts;  3,  sporoblasts  elongated  and  spindle-shaped; 
4,  two  sporozoites  have  developed  in  each  sporoblast;  4  a,  the  cyst  ruptured 
and  the  sporoblasts  and  the  sporozoites  escaping;  5,  epithelial  cell  of  the  in- 
testinal tract;  5  a,  5  6,  5  c,  6,  6  a,  stages  in  development  within  the  cell,  the 
schizont  stage;  7,  schizont  dividing  to  form  merozoiites;  7  a,  cell  and  schizont 
ruptured  and  merozoi'tes  escaping.  These  again  infest  the  epithelial  cells  and 
may  repeat  the  cycle  5-7  a.  Others  produce  the  sexual  stages  8  and  8  a  to  11; 
8,  9,  infection  of  a  cell  with  a  merozoite  and  development  of  the  macrogamete; 
10,  cell  ruptured,  exposing  the  macrogamete;  8  a,  infection  of  a  cell  with  a 
merozoite  and  development  of  the  microgametocyte;  9  a,  formation  of  the 
microgametes;  10  a,  liberation  of  the  microgametes;  11,  fusion  of  the  micro- 
gamete  with  the  macrogamete;  12,  13,  14,  15,  development  of  the  mature 
encysted  coccidium  (Cole,  Hadley,  and  Fitzpatrick). 

including  probably  the  toxic  action  of  microorganisms  growing  on 
the  mucous  membranes,  mechanical    obstruction   to  swallowing 


472 


VETERINARY   BACTERIOLOGY 


and  to  respiration,  and  a  progressive  marasmus,  frequently  deter- 
mined by  intestinal  complications." 

Bacteriological  Diagnosis. — An  examination  of  smears  from  in- 
fected membranes  will  show  developmental  stages  of  the  organism. 
The  cysts  may  usually  be  demonstrated  in  the  feces. 

Transmission. — The  disease  is  undoubtedly  acquired  by  the 
ingestion  of  cysts  which  have  been  given  off  in  the  feces  of  diseased 
fowls.  It  is  believed  probable  that  wild  birds  may  also  become 
infected,  and  aid  materially  in  the  dissemination  of  the  organism 

Coccidium  ctmiculi 

Disease  Produced. — Coccidiosis  of  the  rabbit. 


Fig.  210. — Coccidium  cuniculi:  a,  6,  c,  Schizonts,  with  production  of  mero- 
zoites  which  may  repeat  the  cycle  or  develop  the  sexual  stage;  e,  f,  g,  develop- 
ment of  the  macrogamete;  h,  i,j,  8,  development  of  the  microgametocytes  and 
microgametes;  A;,  mature  coccidium,  which  encysts  and  divides  to  form  four 
sporoblasts;  /,  formation  of  the  sporozoites  and  their  liberation  by  a  rupture 
of  the  cyst  (Schaudinn). 

Rivolta,  in  1878,  first  described  this  organism  from  the  rabbit 
It  occurs  in  the  intestinal  epithelium.     It  may  be  present  without 


SPOROZOA  473 

evidence  of  disease,  but  is  undoubtedly  the  cause  of  serious  epi- 
zootics among  tame  and  wild  rabbits.  It  is  possible  that  this 
species  is  identical  with  the  preceding. 

Coccidium  of  Cattle 

Several  European  investigators  have  described  a  Coccidium 
as  the  cause  of  bloody  feces,  without  fever  and  with  progressive 
emaciation  in  cattle.  The  organisms  in  the  stools  of  infected 
animals  are  18  to  25  by  13  ^.  Infection  experiments  have  been 
successful  in  reproducing  the  disease. 

Coccidium.  of  Sheep 

A  number  of  students  in  the  United  States  and  Europe  have 
reported  a  coccidiosis  of  sheep.  The  symptoms  and  organisms 
are  similar  to  the  preceding. 


CHAPTER  XLIV 

PATHOGENIC  PROTOZOA  OF  THE  INFUSORIA 

THE  Infusoria  are  differentiated  from  other  protozoa  by  the 
presence  of  cilia  at  some  stage  in  the  life-history,  by  the  presence 
usually  of  mouth  parts  for  swallowing  or  sucking,  and  by  the  pres- 
ence of  two  kinds  of  nuclei,  micronuclei  and  macronuclei.  Repro- 
duction is  usually  accomplished  by  simple  fission  or  by  budding. 
Only  a  single  genus  and  a  single  species  is  known  to  be  pathogenic. 

Balantidiwm  colt 

Disease  Produced. — A  rare  fatal  enteritis  in  man. 


Fig.  211. — Balantidium  coli  in  a  blood-vessel  of  the  submuoosa  of  the  intes- 
tines (Bowman,  in  "  Philippine  Journal  of  Science"). 

This  organism  is  oval  in  shape — 50  to  70  by  60  to  100  (* — and 
a  funnel-shaped  peristome  which  terminates  at  the;  mouth. 


474 


PATHOGENIC    PROTOZOA    OF  THE    INFUSORIA  475 

Cilia  cover  the  surface.  The  two  nuclei  and  two  contracted 
vacuoles  may  commonly  be  made  out.  An  excretory  apparatus 
is  evidenced  by  the  extrusion  of  waste  material  at  a  definite  point 
in  the  cell  surface.  The  life-history  is  relatively  complex.  The 
cell  commonly  multiplies  by  direct  division.  Conjugation  and 
encystment  may  occur. 

The  Balantidium  coli  appears  to  be  a  common  inhabitant  of 
the  intestinal  tract  of  swine.  A  large  number  of  instances  are 
recorded  in  which  it  has  been  found  associated  in  man  with  a 
severe  and  even  fatal  type  of  diarrhea.  The  connection  of  this 
organism  with  the  disease  as  a  causal  agent  rather  than  as  a  com- 
mensal seems  to  be  well  authenticated.  It  appears  that  infection 
may  follow  the  ingestion  of  the  cysts  produced  by  the  organism. 
The  disease  has  been  reported  from  Europe,  the  United  States,  and 
the  Philippines. 


SECTION   VI 

INFECTIOUS  DISEASES  IN   WHICH  THE  SPECIFIC 
CAUSE  IS  NOT  CERTAINLY  KNOWN 


CHAPTER  XLV 

DISEASES  PRODUCED  BY  ULTRA-MICROSCOPIC  ORGANISMS 

WITHIN  the  last  two  decades  there  have  been  described  a 
number  of  diseases  in  which  the  causal  organism  is  said  to  be 
ultramicroscopic.  By  this  is  meant  that  with  the  best  powers  of 
the  microscope  no  definite  organism  can  be  distinguished.  Such 
an  organism  is  frequently  called  a  filterable  virus,  because  filtrates 
passed  through  fine-pored  porcelain  filters  retain  their  pathogenicity 
for  susceptible  animals. 

It  is  a  principle  of  optics  that  no  object  can  be  clearly  differen- 
tiated that  is  smaller  than  one-half  the  wave-length  of  the  light 
in  which  it  is  examined.  This  means  that  there  is  an  apparently 
insuperable  physical  obstacle  to  the  observation  of  some  of  these 
forms,  as  our  best  lenses  approach  moderately  near  to  this  limit 
in  their  magnification.  Our  reasons  for  believing  that  there  are 
organisms  this  small  will  be  discussed  under  the  heading  of  various 
diseases. 

Recently  there  has  come  into  use  an  instrument  known  as  the 
ultramicroscope,  which  renders  visible  objects  more  minute  than 
had  heretofore  been  observed.  This  instrument  enables  one  to 
observe  objects  by  making  use  of  the  principle  worked  out  by 
Tyndall  in  determining  the  absence  of  floating  dust-particles  in  the 
air.  He  noted  that  when  a  ray  of  a  very  bright  light  was  admitted 
to  a  darkened  room  or  box,  this  ray  could  be  distinctly  seen  as 
long  as  there  were  any  floating  particles,  but  became  invisible 
when  these  dust-particles  had  completely  subsided.  The  motes  or 

476 


DISEASES   PRODUCED   BY   ULTRA-MICROSCOPIC   ORGANISMS      477 

particles,  even  when  smaller  than  can  usually  be  seen  by  the  un- 
aided eye,  became  visible  when  thus  illuminated.  This  principle  is 
applied  to  microscopic  examination  by  sending  the  rays  from  an 
arc  light  or  similar  source,  so  that  they  are  concentrated  in  a 
powerful  beam,  which  is  passed  through  the  hanging  drop  or  similar 
preparation  from  side  to  side.  Exceedingly  minute  particles  may 
thus  be  made  visible.  The  use  of  this  instrument  has  been  found 
to  be  less  helpful  in  the  fields  of  biological  research  than  had  been 
hoped.  Very  few  facts  concerning  the  organisms  that  cause 
disease,  and  particularly  the  ultra-microscopic  organisms,  have 
been  discovered  by  its  aid. 

An  attempt  has  sometimes  been  made  to  compare  the  relative 
size  of  ultramicroscopic  organisms  by  the  use  of  porcelain  filters 
of  different  degrees  of  density.  It  has  been  found  that  the  virus 
of  some  diseases  will  pass  through  coarse  filters,  but  not  through 
the  finer  ones.  It  does  not  seem  to  be  entirely  a  matter  of  the 
relative  size  of  pores  and  organisms  that  may  pass  through  the 
filter.  It  is  probably  a  phenomenon  analogous  to  an  adsorption 
quite  as  much  as  mechanical  filtration  that  removes  the  organ- 
isms. 

Bacterial  or  Protozoan  Relationships  of  Ultramicroscopic 
Organisms. — There  is  no  practicable  method  telling  certainly 
whether  or  not  an  ultramicroscopic  virus  should  be  grouped  with 
the  protozoa  or  with  the  bacteria.  There  are  methods  which  may 
sometimes  be  used  that  will  give  inferences,  however.  It  has  been 
found  possible  in  some  cases  to  secure  growth  in  culture-media, 
such  as  is  used  for  bacteria.  Such  organisms  are  probably  bac- 
teria. In  others,  the  type  of  disease  produced  may  resemble  so 
closely  some  other  infection  produced  by  a  known  organism  that  a 
probable  classification  into  protozoan  or  bacterial  might  be  made. 
The  type  of  immunity  developed  may  likewise  be  of  importance. 
In  most  instances  these  differences  are  not  pronounced  enough  to 
allow  of  certainty  in  the  classification. 

The  more  important  diseases  which  have  been  described  as 
due  to  ultramicroscopic  organisms  are  contagious  pleuropneumonia 
of  cattle,  rinderpest  or  cattle  plague,  foot-and-mouth  disease,  hog 
cholera,  horse  sickness,  dog  distemper,  fowl  plague,  equine  anemia, 
fowl  pox,  yellow  fever,  and  epidemic  infantile  paralysis. 


478  VETERINARY   BACTERIOLOGY 

Virus  of  Pleoropneomonia 

Disease  Produced. — Pleuropneumonia,  peripneumonia,  lung 
plague  of  cattle  and  other  bovines. 

Nocard  and  Roux  described  the  causal  organism  in  1898.  The 
disease  itself  has  been  known  in  Europe  for  several  centuries. 

Distribution. — The  disease  is  known  from  Europe,  Africa, 
Australia,  Asia,  and  has  been  imported  into  the  United  States, 
where,  in  1886,  it  killed  10,000  animals  in  Illinois  alone. 

Nature  of  Virus. — The  causal  organism  is  doubtless  a  bacterium 
that  is  just  at  the  limit  of  visibility.  In  suitable  fluids  it  may  be 
observed  under  very  high  powers  as  a  tiny  motile  point.  Bouillon 
inoculated  with  the  serous  exudate  from  the  pleura  or  from  one 
of  the  areas  of  consolidation  in  the  lungs,  sealed  in  a  collodion 
sac,  and  placed  in  the  peritoneal  cavity  of  a  rabbit  for  two  weeks, 
will  show  a  slight  clouding  or  opalescence.  Transfers  to  new  media 
similarly  treated  will  likewise  result  in  growth.  This  material 
may  be  shown  to  be  infective.  The  organism  may  also  be  cul- 
tivated in  a  mixture  of  bouillon  and  blood-serum  outside  the  animal 
body.  In  two  to  three  days  it  shows  a  very  faint  clouding.  Agar 
containing  serum  develops  delicate,  transparent,  almost  invisible, 
colonies.  The  optimum  temperature  is  37°;  no  growth  occurs 
under  30°. 

Pathogenesis. — Injection  of  pure  cultures  of  the  organism  into 
cattle  results  in  infection.  Intrapleural  injections  or  inhala- 
tions of  the  organism  result  in  a  typical  clinical  picture  of  the 
disease.  The  disease  is  characterized  by  more  or  less  extensive 
areas  of  hepatization  in  the  lungs  and  an  inflammation  of  the 
pleura,  accompanied  by  a  serofibrinous  exudate.  The  amount  of 
fluid  which  collects  may  be  very  considerable. 

Immunity. — Recovery  from  the  disease  results  in  a  relatively 
permanent  immunity.  Immunization  against  the  disease  by 
vaccination  with  serum  from  the  pleural  cavity  of  infected  animals 
has  been  practised.  The  material  is  injected  subcutaneously. 
Its  use  is  attended  with  danger,  as  from  0.5  to  5  per  cent,  or  even 
more  of  those  vaccinated  have  been  killed  by  the  vaccine.  The 
method  in  question  has  been  of  some  uc.e  in  immunization,  but  a 
stamping-out  process  would  appear  to  be  more  efficacious. 

Nocard  and  Roux  have  advised  vaccination  with  pure  cultures, 


DISEASES   PRODUCED   BY    ULTRA-MICROSCOPIC   ORGANISMS      479 

and  claim  to  have  secured  more  favorable  results  than  by  the 
older  method.  Nocard  has  also  produced  a  curative  and  pro- 
phylactic serum  by  the  hyperimmunization  of  animals  by  the 
injection  of  increasing  doses  of  pure  culture  until  six  liters  have 
been  used.  In  doses  of  40  c.c.  it  served  as  an  efficient  prophylactic, 
and,  in  larger  amounts,  as  a  curative  agent  in  the  early  stages  of  the 
disease. 

Transmission. — The  method  of  natural  spread  of  the  disease 
is  not  certainly  known.  It  is  probably  through  inhalation  of  the 
causal  organism. 

Virus  of  Foot-and-mouth  Disease 

Disease  Produced. — Foot-and-mouth  disease,  aphthous  fever, 
Maul-  and  Klauenseuche  in  cattle  and  other  bovines,  sheep,  goats, 
swine,  deer,  and  occasionally  horses,  dogs,  cats,  and  man. 

The  disease  has  been  known  for  over  a  century  in  Europe. 
Loffler  and  Frosch,  in  1897,  Hecker,  in  1898,  and  others  since 
that  time  have  shown  that  the  virus  of  foot-and-mouth  disease 
may  pass  through  Chamberland  and  Berkefeld  filters,  but  not 
through  the  thicker  Kitasato  filter. 

Distribution. — The  disease  is  known  from  most  of  Europe, 
Asia,  and  Africa.  It  has  been  introduced  several  times  into  the 
United  States,  but  has  been  stamped  out.  It  has  also  been 
reported  from  Argentina. 

Nature  of  the  Virus. — All  efforts  at  observation  and  culture  of 
the  organism  have  failed.  The  facts  given  above  show  it  to  be 
a  filterable  virus  and  probably  ultramicroscopic. 

Pathogenesis. — The  disease  may  be  produced  by  the  inocula- 
tion of  susceptible  animals  with  the  filtrate  through  a  porcelain 
filter.  It  is  characterized  by  an  acute  fever,  the  appearance  of  a 
vesicular  eruption  on  the  mucous  membranes  of  the  mouth,  on 
the  feet  and  between  the  toes.  It  is  commonly  not  fatal,  but  is 
so  contagious,  and  leads  to  such  losses  in  flesh  and  milk,  that  it  is 
among  the  most  feared  of  cattle  diseases. 

Immunity. — Vaccination  by  intentional  infection  of  animals  has 
sometimes  been  practised  in  an  effort  to  "  get  it  over  with  "  as 
quickly  as  possible  when  it  breaks  out  in  a  herd.  An  animal 
recovered  is  relatively  immune.  Such  methods  are  attended  by 


480  VETERINARY   BACTERIOLOGY 

considerable  risk.     An  efficient  and  safe  method  of  immunization 
or  vaccination  has  not  been  developed. 

Transmission. — The  organisms  gain  entrance  through  contact 
of  healthy  animals  with  the  saliva  or  other  secretions  of  an  in- 
fected animal.  They  may  be  transmitted  to  young  animals  or  to 
man  in  milk. 

Virus  of  Rinderpest  or  Cattle  Plague 

Disease  Produced. — Cattle  plague,  rinderpest,  or  contagious 
typhus  in  cattle,  rarely  in  sheep,  goats,  and  camels. 

Nocard,  and  later  Tartakowsky,  observed  that  the  body  fluids 
in  animals  having  this  disease  contained  no  visible  microorgan- 
isms, but  were  infective.  Nicolle  and  Adelbey  established  that 
these  fluids,  if  thinned  with  water,  could  be  passed  through  the 
coarser  Berkefeld  filters,  but  not  through  a  fine-pored  Chamber- 
land,  without  losing  their  virulence. 

Distribution. — The  disease  has  been  reported  from  a  large 
portion  of  the  area  of  Europe.  It  is  endemic  in  southern  Asia, 
is  known  in  the  Philippines,  and  has  caused  great  losses  in  Egypt 
and  in  Southern  Africa.  It  has  not  gained  entrance  to  the  United 
States. 

Character  of  Virus. — It  is  both  ultramicroscopic  and  filterable. 
It  has  not  been  cultivated.  Blood  sealed  hermetically  in  tubes  is 
found  to  retain  its  virulence  for  months.  The  virus  is  destroyed 
by  desiccation  or  by  heating  to  58°  to  60°.  It  may  survive  in 
putrefying  flesh  for  considerable  periods.  It  is  easily  destroyed 
by  disinfectants. 

Pathogenesis. — The  virus  is  present  in  all  the  body  tissues 
and  excretions.  One  one-thousandth  of  a  gram  of  blood  from 
an  infected  animal  at  the  height  of  the  disease  has  been  found 
sufficient  to  reproduce  the  disease  in  a  susceptible  animal.  The 
infection  is  an  acute,  highly  fatal  fever,  in  which  there  are  croupous 
diphtheritic  lesions  of  the  intestinal  tract.  It  is  typically  a  cattle 
disease,  but  occasionally  attacks  other  animals. 

Immunity. — Animals  which  recover  spontaneously  from  the 
disease  are  highly  immune,  and  the  blood  has  some  power  of  pas- 
sive immunization  when  injected  into  another  animal.  Vac- 
cination with  the  nasal  secretion  of  sick  animals  into  the  tails  of 


DISEASES   PRODUCED   BY    ULTRA-MICROSCOPIC    ORGANISMS      481 

others  has  been  practised,  and  has  been  found  in  some  instances  to 
result  in  a  low  mortality.  The  method  has  been  practically- 
abandoned.  Injections  of  the  bile  from  animals  having  the  disease 
has  been  advocated  by  Koch  and  extensively  practised  in  South 
Africa,  with  good  results. 

The  common  method  of  immunization  against  rinderpest  is 
that  developed  by  Kolle  and  Turner.  Animals  which  have  recov- 
ered spontaneously  from  an  infection,  or  that  have  been  im- 
munized by  injections  of  virulent  blood  and  gall,  are  hyper- 
immunized  by  repeated  injections  of  virulent  blood.  The  first 
injection  is  of  a  liter  of  virulent  blood.  After  the  subsidence  of  the 
reaction  an  injection  of  500  c.c.  is  given,  and  later  a  third  injec- 
tion of  a  liter.  A  fourth  injection  may  be  made.  The  blood  is 
drawn  from  the  jugular  vein  at  three  intervals,  a  week  apart. 
Another  injection  of  a  liter  of  virulent  blood  is  given,  and  later 
the  animal  is  again  bled.  The  serum,  in  amounts  of  20  c.c., 
should  protect  an  animal  against  injection  of  1  c.c.  of  virulent 
blood.  An  injection  of  50  to  100  c.c.  of  the  serum  so  secured  will 
protect  an  animal  against  infection  for  a  space  of  2-4  months 
usually.  A  more  permanent  immunity  may  be  established  by  the 
use  of  what  is  termed  the  "  serum  simultaneous  "  method.  The 
animal  is  injected  on  one  side  with  8  to  25  c.c.  of  the  immune  serum, 
and  on  the  other  with  1  c.c.  of  virulent  blood.  Some  animals  react 
by  a  distinct  fever,  others  show  no  effect.  The  latter  are  rendered 
immune  for  several  months  only,  while  the  former  for  much  longer 
periods.  The  blood  of  the  animals  reacting  is  infective  during  the 
period  of  fever.  The  vaccination  mortality  is  about  1  per  cent. 

Transmission. — The  disease  is  readily  transmitted  by  means  of 
soiled  food,  water,  and  by  direct  contact. 

Virus  of  Hog-cholera 

Disease  Produced. — Hog-cholera,  Schweinepest,  swine  fever. 

From  the  time  of  the  researches  of  Salmon  and  Smith  on  this 
disease,  published  in  1885,  until  1904,  the  cause  of  hog-cholera  was 
believed  to  be  the  Bacillus  cholera  suis  (B.  suipestifer) .  In  the 
latter  year  de  Schweinitz  and  Dorset  showed  the  typical  hog- 
cholera  in  the  United  States  to  be  due  to  a  filterable  virus.  This 
has  been  confirmed  by  Hutyra,  Uhlenhuth,  and  others  in  Europe. 
31 


482  VETERINARY    BACTERIOLOGY 

Distribution. — The  disease  is  wide-spread  in  Europe  and  North 
America. 

Nature  of  Virus. — The  organism  causing  hog-cholera  is  an  ultra- 
microscopic  filterable  virus.  It  passes  readily  through  porcelain 
filters.  It  has  never  been  cultivated.  Fluid  material  will  retain 
its  virulence  for  a  period  of  ten  to  fourteen  weeks,  at  least,  when 
kept  at  room-temperatures.  It  is  killed  by  exposure  ,to  60°  to  70° 
for  an  hour.  Desiccation  does  not  destroy  it  at  once,  but  only 
after  a  lapse  of  several  days.  It  may  be  destroyed  by  disinfect- 
ants, but  is  relatively  resistant. 

Pathogenesis. — The  subcutaneous  injection  of  1  to  2  c.c.  of 
filtered  blood-serum  or  body  fluids  results  in  the  production  of  the 
disease.  The  animals  may  die  of  a  very  acute  type  of  the  disease, 
or  it  may  assume  a  chronic  form.  The  acute  cases  generally 
reveal,  on  autopsy,  hyperemia  and  acute  swelling  of  the  internal 
organs,  and  hemorrhages  on  the  serous  and  mucous  membranes, 
and  frequently  a  serous  transudate  into  the  pericardium.  In  the 
more  chronic  type  ulcerated  and  necrotic  areas  are  commonly 
found  in  the  intestines,  together  with  pneumonia. 

The  disease  cannot  be  transferred  to  other  animal  species. 
The  Bacillus  cholerce  suis  is  probably  a  secondary  invader,  but  the 
lesions  produced  by  this  organism  may  in  some  cases  be  of  the 
greatest  importance. 

Immunity. — Animals  that  have  recovered  from  an  infection 
with  hog-cholera  are  thereafter  immune,  and  it  has  been  shown 
that  their  blood  has  some  immunizing  power  when  injected  into 
other  susceptible  individuals. 

Practical  methods  of  immunization  were  developed  through 
the  work  of  Dorset,  McBryde,  and  Niles  in  this  country.  The 
method  devised  by  them  and  commonly  used  is  as  follows:  It  is 
necessary  to  start  with  an  animal  that  has  recovered  from  the 
disease  or  that  has  already  been  immunized.  This  animal  is  then 
hyperimmunized  by  repeated  injections  of  virulent  blood.  The 
latter  must  be  shown  to  be  virulent  by  actual  test  before  it  can 
be  used.  About  1  c.c.  of  the  defibrinated  virulent  blood  is  injected 
for  each  pound  of  body  weight.  A  week  later  an  injection  of  two 
and  one-half  times  as  much  is  made,  and  after  another  week  an 
injection  of  5  c.c.  Subsequent  injections  are  made,  preferably 


DISEASES    PRODUCED    BY    ULTRA-MICROSCOPIC   ORGANISMS      483 

intravenously.  The  animal  thus  hyperimmunized  is  bled,  from 
the  tip  of  the  tail  usually,  the  blood  allowed  to  clot,  and  the  serum 
used  for  immunization.  It  is  customary  to  preserve  this  serum 
from  consecutive  bleedings  until  a  considerable  quantity  has  ac- 
cumulated, when  it  is  tested  for  potency.  For  testing  it  is  cus- 
tomary to  use  young  pigs,  weighing  from  30  to  60  pounds.  A 
serum  of  satisfactory  potency  should  protect,  in  a  dose  of  15  c.c. 
or  less,  one  of  these  animals  against  an  injection  of  2  c.c.  of  virulent 
blood.  The  serum  injections  of  20  c.c.  are  commonly  used  to 
protect  pigs  against  infection.  If  the  animals  are  exposed  to  in- 
fection, they  may  have  a  light  attack  of  the  disease,  and  are  there- 
by rendered  permanently  immune.  Where  it  is  desired  to  im- 
munize, and  exposure  to  infection  is  not  certain,  a  much  more 
lasting  .immunity  is  conferred  by  the  use  of  the  serum  simul- 
taneous method.  In  this  method  virulent  blood  (2  c.c.)  is  in- 
jected at  the  same  time  as  the  immune  serum.  This  results  in 
the  development  of  an  active  immunity,  which  is  relatively  per- 
manent in  comparison  with  the  immunity  of  two  to  three  weeks 
conferred  by  antiserum  injection  alone.  The  use  of  this  serum  has 
been  found  to  be  highly  successful  in  practice. 

It  is  not  known  what  property  of  the  antiserum  is  thus  effec- 
tive, whether  it  is  antitoxic,  opsonic,  or  bactericidal.  There  is 
some  evidence  that  it  is  the  last,  but  proof  is  difficult  to  secure. 

Transmission. — The  virus  may  be  demonstrated  in  the  blood, 
the  tissues,  and  the  urine  of  infected  animals.  It  is  probable  that 
infection  commonly  takes  place  through  ingestion. 

Virus  of  Horse  Sickness 

Disease  Produced. — African  horse  sickness,  or  Pferderpest. 

This  disease,  known  from  southern  Africa  for  more  than  a 
century,  was  first  shown  by  MacFadyean  in  1900,  and  later — in 
1901 — by  Xocard,  to  be  due  to  an  ultramicroscopic,  filterable  virus. 
The  disease  is  characterized  as  an  acute  or  subacute  disease  of 
solipeds,  that  appears  in  epizootics  during  the  hot  months  of  the 
year.  The  principal  lesions  are  edematous  swellings  and  hemor- 
rhages of  the  internal  organs.  The  virus  will  pass  through  a 
Berkefeld  or  a  Chamberland  porcelain  filter  if  the  serum  is  diluted 
with  physiological  salt  solution. 


484  VETERINARY    B ACTERIOLC  >< ;  V 

Immunization  against  the  disease  may  be  brought  about  by  the 
use  of  serum  from  hyperimmunized  animals.  Koch  hyperim- 
munized  horses  that  had  recovered  from  the  disease  by  three  to  four 
injections  of  virulent  blood  at  intervals  of  a  week,  as  much  as  two 
liters  being  used  for  the  last  injection.  Serum  simultaneous 
injections  of  this  hyperimmunized  serum  and  virulent  blood 
into  susceptible  animals  in  correctly  proportioned  doses  will 
immunize. 

The  disease  has  been  found  to  be  contracted  generally  at  night, 
and  the  first  frost  puts  an  end  to  the  epizootic  for  the  year.  Con- 
siderable quantities  of  the  virus  must  be  fed  before  an  infection 
is  produced,  showing  that  natural  infection  is  probably  in  some 
other  manner  than  by  ingestion.  It  is  probable  that  mosquitos, 
possibly  flies,  act  as  carriers.  The  disease  cannot  be  regarded, 
therefore,  in  the  strictest  sense  as  contagious. 

Virus  of  Infectious  Anemia  of  the  Horse 

Disease  Produced. — Infectious  anemia,  pernicious  anemia,  mud 
fever,  swamp  fever  of  the  horse. 

This  disease  has  been  known  as  a  clinical  entity  in  Europe  for 
three-quarters  of  a  century.  Carre  and  Villee  (1904-1906)  and 
Ostertag  and  Marek  (1907)  have  demonstrated  the  disease  to  bo 
due  to  a  filterable  virus.  The  disease  has  been  studied  in  North 
America  by  several  investigators,  and  the  ultramicroscopic  nature 
of  the  virus  has  been  independently  demonstrated.  It  is  not 
certain  that  all  the  infections  described  under  this  name  are  iden- 
tical, but  there  is  considerable  evidence  tending  to  establish  such 
as  a  fact. 

Distribution. — The  disease  is  probably  wide-spread,  but  has  not 
always  been  clearly  differentiated.  It  is  known  from  Germany, 
France,  Hungary,  Switzerland,  and  Sweden  in  Europe,  and  from 
Saskatchewan,  Manitoba,  Minnesota,  the  Dakotas,  Nebraska, 
Kansas,  Colorado,  Wyoming,  Montana,  Texas,  and  Nevada  in  the 
United  States. 

Nature  of  the  Virus. — The  causal  organism  is  an  ultramicro- 
scopic,  filterable  virus  that  cannot  be  difTon-ntiatod  by  staii  ing 
methods  and  has  not  boon  cultivated.  It  is  found  in  tho  blood, 
the  urine,  and  the  feces  of  infected  animals.  It  is  destroyed  at  a 


DISEASES   PRODUCED    BY    ULTRA-MICROSCOPIC   ORGANISMS      485 

temperature  of  58°.  It  will  withstand  drying  for  several  months, 
and  liquids  maintain  their  infectivity  for  months,  even  when  decay- 
ing. 

Pathogenesis. — The  virulent  blood  or  blood-serum  will  infect 
another  animal  upon  subcutaneous  injections  of  small  quantities. 
The  incubation  period  after  injection  varies  from  five  to  nine  days, 
or  even  more.  The  initial  symptom  is  a  fever.  The  disease  is 
more  apt  to  be  acute  when  the  organism  is  introduced  by  injec- 
tions than  by  ingestion.  The  disease  may  be  characterized  as  an 
acute  or  chronic  anemia  which  has  the  appearance  of  a  septicemia 
in  which  there  is  a  great  destruction  of  blood-elements.  The 
anatomical  findings  are  characteristic.  Mack,  in  his  work  on 
cases  in  Nevada,  notes  profound  cardiac  and  respiratory  disturb- 
ances. There  is  a  progressive  destruction  of  the  red  blood-cells, 
parenchymatous  degeneration  of  the  kidneys  and  liver,  and  ex- 
tensive changes  in  the  vascular  system.  The  spleen  is  engorged 
and  frequently  degenerated,  and  the  bone-marrow  undergoes  pro- 
found degeneration. 

Immunity.— Xo  method  of  immunization  has  been  developed. 

Transmission. — European  writers  are  of  the  opinion  that  in- 
fection arises  through  the  ingestion  of  food  soiled  by  excretions 
of  infected  animals.  The  mode  of  dissemination  has  not  been 
satisfactorily  established. 

Virus  of  Dog  Distemper 

Disease  Produced. — Dog  distemper,  Hundstaupe. 

Bacteria  belonging  to  several  different  groups,  particularly 
to  the  colon-typhoid  and  to  the  hemorrhagic  septicemia,  have 
been  described  as  the  cause  of  dog  distemper.  Carre,  in  1905, 
attributed  the  cause  to  a  filterable  virus. 

Distribution. — Europe  and  America,  probably  in  other  parts 
of  the  world. 

Nature  of  the  Virus. — Little  is  known  of  the  virus  beyond  the 
fact  that  it  can  be  passed  through  a  porcelain  filter.  It  has  not 
been  cultivated  and  is  probably  ultramicroscopic. 

Pathogenesis.— The  disease  is  a  highly  fatal,  acute  infection 
of  young  carnivorous  animals,  characterized  by  an  acute  catarrh 
of  the  mucous  membrane,  and  frequently  a  catarrhal  pneumonia. 


486  VETERINARY   BACTERIOLOGY 

In  a  small  percentage  of  the  cases  nervous  symptoms  develop. 
The  discharge  from  the  mucous  membranes  is  highly  infective. 
Secondary  infection  with  bacteria  is  common,  and  is  believed  by 
some  investigators  to  account  for  many  of  the  deaths. 

Immunity. — Many  attempts  have  been  made  to  prepare  an  anti- 
or  immune  serum  that  would  be  efficacious  in  preventing  or  curing 
the  disease.  Several  have  been  placed  upon  the  market,  but  none 
has  been  shown  to  be  efficacious.  It  is  possible  that  success 
might  be  attained  by  the  use  of  hyperimmune  blood. 

Transmission. — The  disease  is  transmitted  by  direct  or  indirect 
contact  with  infected  individuals. 

Virus  of  Fowl  Plague 

Disease  'Produced. — Fowl  plague,  chickenpest  of  domestic 
fowls. 

This  disease  has  generally  been  confused  with  chicken  cholera, 
which  it  closely  resembles  clinically,  although  Penoncito  un- 
doubtedly described  it  in  1878.  Centanni  and  Savonuzzi,  in  1901, 
showed  that  the  virus  could  pass  through  a  porcelain  filter.  This 
has  been  amply  confirmed  by  other  writers. 

Distribution. — The  disease  is  known  only  from  northern  Italy, 
the  Tyrol,  Germany,  and  France. 

Character  of  the  Virus. — The  organism  is  a  filterable  virus,  and 
is  probably  ultramicroscopic,  although  Rosenthal,  Kleine,  and 
Schiffman  have  described  bodies  in  the  nerve-centers  that  are 
possibly  protozoan  in  nature.  The  virus  is  found  in  the  blood,  the 
nasal  secretion,  and  generally  throughout  the  tissues.  The  blood 
retains  its  virulence  for  three  months  when  sealed  in  tubes  and 
kept  in  a  dark  place.  The  thermal  death-point  is  55°  for  thirty 
minutes  or  60°  for  five  minutes.  The  dried  virus  has  been  found 
to  retain  its  virulence  for  two  hundred  days,  and  in  glycerin  and 
serum  mixture  for  two  hundred  and  seventy  days.  It  is  easily 
destroyed  by  disinfectants. 

Pathogenesis. — Injection  of  as  small  amount  as  [^55  c-c-  of 
virulent  blood  or  secretions  is  sufficient  to  infect.  The  virus 
is  pathogenic  for  many  birds  besides  fowls,  but  not  for  mam- 
mals. The  disease  is  characterized  by  the  hemorrhages  into 
the  serous  membranes  in  acute  cases,  and  in  the  less  acute  edema 


DISEASES   PRODUCED   BY    ULTRA-MICROSCOPIC   ORGANISMS      487 

of  the  subcutaneous  tissues  and  of  the  serous  membranes,  and  the 
formation  of  a  fibrous  exudate  upon  the  latter. 

Immunity. — Practicable  methods  of  immunization  have  not 
been  developed. 

Transmission. — The  disease  is  transmitted  by  the  ingestion  of 
food  soiled  with  the  infective  feces,  nasal  secretion,  or  blood  of  in- 
fected birds. 

Virus  of  Epithelioma  Contagiosum 

Disease  Produced. — Fowl  pox,  epithelioma  contagiosum  in 
domestic  fowls,  sore  head. 

Marx  and  Sticker,  in  1902,  determined  the  cause  of  fowl  pox 
to  be  a  filterable  virus.  Several  other  investigators  subsequently 
confirmed  their  results. 

Distribution. — The  disease  is  known  to  occur  in  Europe,  in  the 
United  States,  particularly  the  south,  in  California,  and  Hawaii. 

Nature  of  the  Virus. — Marx  and  Sticker  showed  that  when  an 
epithelial  nodule  was  triturated  in  physiological  salt  solution  that 
the  fluid  which  passed  through  a  Berkefeld  filter  was  infective. 
Some  investigators  have  observed  tiny  spherical  granules,  less  than 
0.25  u.  in  diameter,  in  the  emulsion  of  the  virus,  but  it  is  by  no 
means  certain  that  these  are  the  disease-producing  organisms.  The 
virus  is  relatively  resistant  to  unfavorable  conditions.  The 
nodules  may  be  dried  for  weeks  without  losing  their  infectivity. 
It  is  destroyed  by  heating  to  60°  for  eight  minutes.  Mixed  with 
glycerin,  it  retains  its  infectivity  for  many  weeks.  It  is  easily 
destroyed  by  disinfectants. 

Pathogenesis. — The  disease  is  a  chronic,  contagious  infection, 
characterized  by  an  initial  catarrh  of  the  mucosa  of  the  head, 
followed  by  wart-like  growths  (epithelial  hyperplasia)  of  the  skin, 
especially  of  the  comb  and  naked  skin  of  the  head,  sometimes 
associated  with  a  croupous  diphtheritic  condition  of  the  mucosa 
of  the  head.  This  latter  condition  is  one  of  those  grouped  under 
the  general  name  of  fowl  diphtheria.  The  disease  commonly 
terminates  favorably  in  three  to  five  weeks. 

Immunity. — No  practicable  method  of  immunization  has  been 
devised. 

Transmission. — The  disease  is  transmitted  by  direct  contact 
with  infected  fowls. 


488  VETERINARY   BACTERIOLOGY 

Virus  of   the  Poxes 

Disease  Produced. — Small-pox  in  man,  cow-pox,  sheep-pox, 
horse-pox,  swine-pox,  goat-pox. 

There  is  much  doubt  relative  to  the  position  of  the  virus  of  the 
various  poxes.  They  are  included  in  this  group  tentatively,  as  it 
is  found  that  the  contents  of  the  vesicles  of  the  eruptions  may  be 
filtered  through  a  thin,  coarse  porcelain  filter  under  pressure  with- 
out losing  their  infectivity.  The  causal  microorganisms  are,  at 
some  stages,  at  least,  filterable  and  probably  ultramicroscopic. 
Certain  cell  inclusions  have  been  described  as  being  probably  of 
protozoan  nature,  but  the  subject  cannot  be  said  at  present  to  be 
completely  elucidated.  The  protozoan  parasite  has  been  named 
Cytorhyctes  vaccince  in  man. 

Immunity. — Recovery  from  an  attack  of  variola  is  accompanied 
by  a  relatively  permanent  immunity.  Vaccination  is,  therefore, 
commonly  practised,  particularly  against  small-pox  in  man.  The 
attenuated  virus  in  this  instance  is  secured  by  passage  through 
an  animal,  usually  a  cow.  The  vaccinating  material  is  the  lymph 
from  the  vesicles  produced  on  the  animals.  It  is  inoculated  into 
the  skin  by  scarification.  The  virulence  is  apparently  very 
greatly  decreased  by  this  method  of  inoculation,  so  that  a  relatively 
mild  type  of  disease  is  produced  which  terminates  in  immunity 
being  established.  To  what  this  immunity  may  be  due  is  not 
known. 

Virus  of  Yellow  Fever 

Yellow  fever  in  man  has  been  shown  to  be  due  to  a  filterable 
virus,  probably  an  ultramicroscopic  organism.  All  efforts  at 
cultivation  have  failed.  The  disease  is  spread  only  through  the 
bite  of  mosquitos  that  have  taken  virulent  blood.  The  organ- 
ism evidently  undergoes  a  part  of  its  life-cycle  in  the  blood  of  the 
mosquito  (Stegomyia),  for  the  latter  does  not  become  infective 
itself  for  several  days.  It  is  evidently  more  than  a  mere 
mechanical  transfer  of  the  organism  by  the  mosquito;  the  latter 
serves  as  a  true  intermediate  host. 

Virus  of  Epidemic  Infantile  Paralysis 

Disease  Produced. — Acute  poliomyelitis,  Il<  ine-Medin  disease 
in  children. 


DISEASES   PRODUCED    BY    ULTRA-MICROSCOPIC    OUCJAMSMS 

This  disease  is  known  from  Sweden,  Germany,  and  the  United 
States.  Flexner  and  Lewis,  in  1909,  have  shown  the  organism  to 
be  a  filterable,  probably  ultramicroscopic,  virus.  The  disea>e 
may  be  transferred  to  the  monkey.  It  probably  spreads  by  inges- 
tion  of  infected  materials. 

Virus  of  Rabies 

Disease  Produced. — Rabies  in  animals.  Hydrophobia  in  man. 
Lyssa. 

The  disease  has  been  studied  at  great  length  by  many  in- 
vestigators, and  there  is  still  great  disparity  of  opinion  as  to  the 
nature  of  the  cause.  Remlinger  and  Riff  at  Bey,  in  1903,  showed 
that  the  virus  could  be  passed  through  a  porous  Berkefeld  filter. 
This  has  been  substantiated  since  by  several  workers.  As  will 
be  seen  below,  this  does  not  satisfactorily  settle  the  problem,  as 
those  who  hold  to  the  protozoan  nature  of  certain  bodies  in  the 
nerve-centers  in  the  disease,  as  they  contend  that  extremely 
minute  plastic  stages  in  the  life-cycle  of  the  organism  might  easily 
pass  through. 

Distribution. — The  disease  is  world  wide  in  distribution. 

Nature  of  the  Virus. — Students  of  the  etiology  of  this  disease 
may  be  divided  into  two  groups — those  who  believe  in  the  presence 
of  a  specific  ultramicroscopic  organism,  and  those  who  believe 
in  the  presence  of  a  protozoan  with  certain  stages  of  development, 
when  the  organism  is  small  enough  to  pass  the  pores  of  the  filter. 
The  latter  theory  has  been  developed  by  Negri.  The  organism  has 
been  named  Neuroryctes  hydrophobia.  In  1903  he  demonstrated 
the  presence  of  specific  bodies,  which  have  been  termed  Xegri 
bodies,  in  the  larger  ganglia-cells  of  the  Ammon's  horn,  as  well  as 
in  other  parts  of  the  central  nervous  system.  There  is  little  ques- 
tion but  what  these  bodies  are  characteristic  of  the  disease:  the 
disputed  point  is  whether  they  are  specific  organisms  or  degenera- 
tion products  of  the  cell.  Williams  and  Lowden  summarize  the 
evidence  of  the  protozoan  nature  of  these  organisms  as  follows : 

"  They  have  definite  characteristic  morphology;  this  mor- 
phology is  constantly  cyclic,  i.  e.,  certain  forms  always  predominate 
in  certain  stages  of  the  disease,  and  a  definite  series  of  forms  in- 
dicating growth  and  multiplication  can  be  demonstrated;  the 


490 


VETERINARY   BACTERIOLOGY 


structure  and  staining  qualities,  as  shown  especially  by  the  smear 
method  of  examination,  resemble  that  of  certain  known  protozoa, 
notably  of  those  belonging  to  the  suborder  Microsporidia." 

The  Negri  bodies  in  suitably  stained  preparations  are  found 
to  vary  in  size  from  less  than  0.5  to  25  p.  In  shape  they  may  bo 
spherical,  ovoid,  or  ellipsoidal.  The  bodies  show  a  characteristic 
structure,  a  smooth  hyaline  margin,  with  inclusions  of  various 
kinds  that  resemble  chromatin  granules. 

They  may  be  readily  stained  by  Giemsa's  method,  or  with 
eosin  and  methylene-blue. 


Fig.  212. — A  Negri  body.     Note  the  circle  of  chromatoid  granules  about  the 
central  body  (X  2000)  (Williams  and  Lowden). 

Pathogenesis. — The  organism  enters  the  body  through  wounds, 
usually  bites  of  animals.  It  then  passes  slowly  along  the  per- 
ipheral nerves  to  the  central  nervous  system.  The  portion  of 
this  to  which  these  nerves  directly  lead  is  the  most  seriously 
affected.  Characteristic  gross  anatomical  lesions  are  quite  lacking 
in  this  disease. 

The  period  of  incubation  is  variable;  it  is  usually  several 
weeks.  It  probably  represents  the  period  necessary  for  the  virus 
to  reach  the  central  nervous  system  and  develop  there.  The  dis- 
ease is  commonly  fatal.  It  affects  most  mammals,  including  man, 
but  is  primarily  a  disease  of  the  carnivora,  particularly  the  dog. 

Immunity.— The  Pasteur  method  of  treatment  is  essentially  a 


DISEASES   PROIHTKD    BY    UI/TRA-MICROSCOPIC   ORGANISMS      491 

method  of  vaccination  at  intervals  with  attenuated  virus.  The 
virus  is  found,  on  experimentation,  to  be  rather  variable  in  its 
power  to  produce  disease.  The  virulence  is  exalted  by  repeated 
inoculations  of  rabbits  until  it  becomes  the  "  fixed  "  virus  of 
Pasteur,  and  will  kill  rabbits  in  six  to  seven  days.  This  is  then 
injected  into  a  rabbit,  and  upon  its  death  the  spinal  cord  is  care- 
fully removed  with  all  aseptic  precautions,  and  suspended  in  a 
desiccator  over  caustic  potash.  It  is  kept  at  a  constant  tempera- 
ture of  23°  in  the  absence  of  light  for  two  weeks.  The  vaccine 
consists  of  an  emulsion  of  this  cord  in  physiological  salt  solution. 


Fig.  213. — Removal  of  the  spinal  cord  from  a  rabbit  (Stimson,  Bull.  Xo.  65, 
Hygienic  Laboratory). 

Later,  injections  are  made  with  a  cord  that  has  been  dried  for  a 
shorter  period.  Repeated  injections  are  made.  The  fact  that  the 
disease  has  normally  a  long  incubation  period  gives  an  oppor- 
tunity in  the  human  for  the  use  of  this  method.  The  active 
immunity  established  by  the  injection  of  the  attenuated  virus  is 
sufficient  to  destroy  the  infecting  organism.  This  method  of 
treatment  has  been  highly  successful  when  commenced  in  time. 
It  is  still  strictly  an  active  immunity.  To  what  principle  it  is  due 
is  not  known. 

Bacteriological  Diagnosis. — The  disease  may  be  diagnosed  by 


492  VETERINARY    BACTERIOLOGY 

animal  inoculation  and  by  macroscopic  examination.  For  the 
former  it  is  customary  to  inject  an  emulsion  from  the  brain  into  a 
rabbit.  The  inoculation  is  usually  made  subdurally.  Sections 
or  smears  may  be  made  from  the  brain  and  stained  to  show  the 
characteristic  Negri  bodies.  Small  portions  of  the  gray  substance 
are  removed  from  the  cerebral  cortex  in  the  region  of  the  crucial 
sulcus,  the  cerebellar  cortex,  and  the  hippocampus  major.  These 
are  crushed  on  a  slide  and  a  smear  made  by  means  of  a  cover-glass. 
These  dried  smears  may  be  stained  by  Giemsa  or  other  stains, 


Fig.  214. — Method  of  drying  the  spinal  cord  of  a  rabbit  for  the  purpose  of 
attenuation  (Stimson,  Bull.  No.  65,  Hygienic  Laboratory). 

perhaps  most  readily  by  the  method  described  by  Williams  and 
Lowden:  "To  10  c.c.  of  distilled  water  three  drops  of  a  saturated 
alcoholic  solution  of  basic  fuchsin  and  2  c.c.  of  Loffler's  solution  of 
iiH-thylcnc-Mur  arc  added.  The  smears  are  fixed  while  moist  in 
methyl  alcohol  for  one  minute.  The  stain  is  then  poured  on, 
warmed  till  it  steams,  poured  off,  and  the  smear  is  rinsed  in  water 
and  allowed  to  dry." 

Transmission. — The  saliva  of  diseased  animals  is  found  to  be 
infective,  and  the  disease  is  transmitted  commonly  through  the  bite. 


BIBLIOGRAPHICAL    INDEX 


ADELBERG,  480 
Arloing,  355 
Arning,  328 
Arthus,  176 
Ayers,  206 

BABES,  258,  260,  456,  460 

Bail,  181,  335,  337 

Balfour,  450 

Bang,  324,  341,  343,  344,  345 

Banzhaf,  142,  143 

Barber,  107 

Bateman,  425 

Battaglio,  425 

Behring,  23,  138 

Berg,  410 

Beurmann,  405 

Blanchard,  440 

Blencig,  21 

Bloch,  407 

Bellinger,  302,  375 

Bolton,  272 

Bowman,  474 

Bredini,  427 

Brodin,  433 

Bruce,  221,  425,  429 

Buckley,  269,  270 

Bumhall,  302 

Bumm,  224 

Busse,  386 

CALKINS,  414 
Calmette,  323,  324 
Carini,  425 
Carre,  484,  485 
Castellani,  454 
Cazalbou,  4:;:i 
Centanni,  4M> 
Chagas,  437 


Chamberland,  336 
Charrin,  238 
Chaussat,  437 
Clegg,  377,  417,  418 
Cohn,  20 
Cole,  471 
Conradi,  284 
Corda,  372 
Cornevin,  355 
Councilman,  420 
Craig,  416,  418,  420 

DANMAN,  210 

Danysz,  276 

Davaine,  22,  331 

de  Beurmann,  40"> 

Deneke,  371 

Denys,  319 

de  Schweinitz,  271,  481 

Desmon,  158 

Dodd,  451 

Doerr,  284 

Dorset,  95,  271,  272,  481,  482 

Douglas,  167 

Dutton,  432,  436,  446 

Duval,  446 

EBERTH,  278 

Ehrenberg,  20 

Ehrlich,  23,  129,  131,  138,  139,  158 

Eichhorn,  256 

Elmassian,  431 

Emmerich,  263 

Emmerlich,  247 

Emmet,  395 

Engler  and  Prantl,  76 

Eppinger,  382 

Escherich,  263,  266 

Evans,  22,  42:5,  42s 

493 


494 


BIBLIOGRAPHICAL   INDEX 


FEHLEISEN,  197 

Femmore,  302 

Kinkier,  371 

Fit /put  rick,  471 

Flexner,  220,  282,  489 

Foth,  259 

Frankel,  215,  267 

Fraser,  429 

Friedlander,  215,  267 

Frosch,  479 

Frost,  97 

Frost  and  McCampbell,  76 

Frothingham,  326,  405 

GABRITSCHEWSKY,  448 
Gaffky,  278 
Galli-Valerio,  384,  461 
Gamaleia,  367,  369 
Gartner,  268 
Gerber,  441 
Gessard,  228 
Gibson,  142 
Gilchrist,  386 
Gonder,  428 
Graham-Smith,  461 
Grassberger,  355,  357 
Gruby,  408 
Guglienni,  458 
Guinard,  238 
Gwyn,  274 

HADLEY,  277,  278,  470,  471 

Haffkine,  306 

Hansen,  328 

Harris,  338,  339,  340 

Hartmann,  416,  422 

Harvey,  277 

liar/,  375 

Haslam,  398 

Hecker,  212,  479 

Heinemann,  204,  205,  206 

Ih-ktoen,  203,  204 

Henle,  22 

Hertel,  298 

Hess,  212 

Hill,  99 

II  r^-hf elder,  319 

282 
Hoffmann,  237,    l.'.l 


Hueppe,  293 
Hutyra,  481 

JOBLING,  220 

Johnes,  220,  226,  326 

Johnson,  206 

Jordan,  338,  339,  340,  372 

KARLIXSKI,  200 

Kartulis,  419 

Kirkpatrick,  278 

Kitasato,  303,  349,  350,  355 

Kitt,    196,   246,  250,  298,  302,  357, 

408 

Klebs,  231,  319 
Kleine,  425,  429,  431,  486 
Knapp,  442,  447,  450 
Koch,  99,  248,  309,  319,  331,  359,  369, 

419,  458,  481,  484 
Koidsumi,  416 
Kolle,  306,  481 
Konew,  255 
Koram,  284 
Krai,  408 

Krumweide,  325,  326 
Kruse,  204 
Klihne,  254 
Kumbein,  306 
Kutscher,  260 

LAFLEUR,  420 

Lafosse,  207 

Lambl,  418 

Landmann,  319 

Lange,  247 

Laveran,  22,  433,  435,  462 

Leclainche,  247 

Leeuwenhoek,  18,  20 

Leger,  437,  467 

Irishman,  204,  438,  439,  441,  447 

Levaditi,  449 

L<-\vis,  423,  437,   Ivi 

LirbiK,  21 

Ugim-res,  293,  297,  298,  :•;•_': J 

Lister,  24 

Loesch,  419 

LofH.-r,  95,  232,  237,  244,  250,  276, 

298,  345,  479 
Lorenz,  247 


BIBLIOGRAPHICAL   INDEX 


495 


Lowden,  489 
Lucet,  200 

MACFADYEAX,    4^3 

Mack,  210,  485 

MacXeal,  426,  430 

Mallasez,  23s 

Manengold,  210 

Marchoix,  442,  448,  450 

Marck,  484 

Mannorek,  203 

Marx,  247,  487 

Mastbaum,  247 

Mathis,  437,  467 

Mayer,  215,  298,  395 

McBryde,  272,  482 

McCampbell,  171,  173,  362,  364,  462 

McCampbell  and  Frost,  76 

McFarland,  174 

McGell,  446 

Melvin,  402 

MetchnikofT,  23,  126,  166,  185 

Micellone,  226 

Migula,  20,  76 

Milne,  446 

Mohler,  210,  222,  256,  269,  270,  346, 

347,  348,  402 

Moore,  201,  210,  266,  314,  427 
Morse,  277,  346,  348 
Miiller,  F.,  20 
Murchison,  23 
Musgrave,  377,  417,  418 

XEGRI,  489 

Xeisser,  161,  224,  328 

Nichols,  389 

Nicolaier,  349 

Xicolle,  439,  480 

Xiles,  482 

Xocard,  197,  238,  241,  242,  266,  276, 

378,  478,  479,  480 
Xorgaard,  210,  238,  402 
Xovy,  426,  430,  442,  447,  450 
Xowak,  341,  344 
Xuttall,  154,  461,  467 

OBERMEIER,  444 
Ogston,  192,  197 


i  Ostertag,  211,  213,  220,  301,  484 
Otho,  283 

PAGE,  405 

Paige,  405 

Park,  325,  326 

Parum,  166 

Pasteur,  21,  22,  197,  244,  246,  295, 

297,  331,  335,  336,  359 
Patton,  456,  462 
Pecaud,  433,  435 
Perkins,  404 
Perroncito,  295,  486 
Pfeiffer,  157 
Pfuhl,  367 
Phalen,  389 
Phillips,  462 
Pirquet,  von,  323 
Polk,  377 
Pollender,  331 
Pong,  446 
Porrey,  226 
Posades,  389 
Prani,  461 

Prantl  and  Engler,  76 
Preisz,  238 
Prettner,  247 
Prior,  371 
Prowazek,  441 

RABE,  226 
Raebiger,  212 
Redi,  20 
Rees,  463 
Remlinger,  489 
Rettger,  277,  278 
Reynolds,  302 
Ricketts,  121 
Rivolta,  226,  384,  472 
Robin,  130 
Rodenwalt,  425 
Rodet,  430 
Roger,  238 
Rosenau,  277 
Rosenbach,  192,  196,  197 
Rosenthal,  284,  486 
Ross,  446 
Rouget,  426 


496 


BIBLIOGRAPHICAL    INDEX 


Roux,  232,  258,  336,  478 
Ruediger,  203 

SACHAROFF,  448 

Salimbeni,  448,  4,50 

Salmon,  271,  481 

Savonuzzi,  486 

Schattenfroh,  355,  3~>7 

Schaudinn,  416,  418,  425,  451 

Schenk,  404 

Schiffman,  486 

Schreiber,  298 

Schreuber,  247 

Schubert,  247,  256 

Schiitz,  207,  247,  250,  256,  298,  345 

de  Schweinitz,  271,  481 

Seller,  260 

Shiga,  282 

Sieber,  428,  457 

Silberschmidt,  380 

Smith,  271,  299 

Smith,  T.,  312,  314,  351,  456,  481 

Sobernheim,  338 

Solleysel,  207 

Starcovici,  456 

Sternberg,  215 

Sticker,  487 

Stimson,  491 

Spitz,  298 

Symons,  429 

TAKTAKOWSKY,  480 

Thrilcr,  430,  435,  436,-  450,  458,  465 

Thom:i>.  :;.").").  427 

Thuillf-r,  244,  246 

Todd,  209,  2S4,  432,  446,  447 

Tokoshige,  384,  385,  386 

Toussant,  331 

Trevisan,  293 


Turner,  481 
Tyndall,  476 

UHLENHUTH,  155,  481 
Uschinsky,  94 

VALENTINE,  423 
Vallet,  430 
Van  Ermengem,  364 
Veyl,  349 
Viereck,  422 
Vignal,  238 
Villee,  484 
Villemin,  309 
Vincent,  381 
Vladimiroff,  258 
Voges,  247,  431 
von  Behring,  23,  138 
von  Pirquet,  323 

WASHBURN,  222,  347 
Wassermann,  230,  301 
Watson,  468 
Webber,  402 
Weichselbaum,  218,  250 
Weigert,  22,  133 
Weil,  300 
Welch,  361 
Werner,  422 
Werneke,  389 
Wesbrook,  233 
Wherry,  328 
Wichsberg,  161 
Widal,  407 
Williams,  489 
Wilson,  302 
Wright,  167,  376,  439 

YERSIN,  232,  303 


INDEX 


ABORTION  bacillus,  341 
group,  189,  341 

contagious,  in  mares,  213 
Abrin,  130 
Abrus  precatorius  as  a  source  of  toxin, 

130 

Absorption  of  complement,  164 
Acanthia  lectularia,  445 
Acetic  acid  fermentation,  64 
Achorion,  393,  408 

schoenleinii,  409 
Acid,  acetic,  64 

butyric,  64 

fermentation,  62 

lactic,  63 

production,  tests  for,  96 
Acid-fast     bacteria,    non-pathogenic, 
329 

group,  188,  308 

organisms,  staining  of,  104 
Acquired  immunity,  122 
Actinomyces,  77,  81,  372 

bovis,  374,  375 

caprae,  374,  380 

coelicolor,  374 

cuniculi,  345 

eppingeri,  374,  382 

farcinica,  378 

group,  189,  372 

madura?,  374,  381 

nocardii,  374,  378 
Actinomycosis  of  cattle,  375 

of  dogs,  380 

of  goats,  380 
Active  anaphylaxis,  178 

immunity,  22,  123 

methods  of  conferring,  123 
Addiment,  159 
Aerobic,  47 
32 


Aerotaxy,  50 

African  horse  sickness,  4>:; 

Agar,  nutrient,  93 

plate  cultures,  113 

stroke  cultures,  10 
Agglutination,  128,  147 

group,  150 

macroscopic  test,  153 

microscopic  test,  152 

tests  for  diseases,  151 
Agglutinin,  147 

body,  149 

chief,  150 

coagglutinin,  150 

constitution,   148 

Ehrlich's  theory  of  production,  148 

flagellar,  149 

group,  150 

hemagglutinins,  153 

immune,  147 

normal,  147 

significance  in  immunity,  153 

somatic,  149 
Agglutinogen,  148 
Agglutinoid,  149 
Agglutinophore,  149 
Aggressins,  181 
Alcoholic  fermentation,  62 
Alexin,  158 
Algae,  26 

Alkali  production,  tests  for,  96 
Alkali-poisoning,  338 
Allergin,  177 
Alpha-amido-acids,  65 
Alt  tuberculin,  318,  321 
Amanita  as  source  of  toxin,  130 
Amboceptor,  158 

action  of,  158 

specificity,  158 

497 


498 


INDEX 


Amboceptor,  structure,  158,  159 
Amebic  dysentery,  419 
Ameboid  colony,  113 
Ammonia,  oxidation,  69 

tests  for,  96 
Amceba,  416 

coli,  416,  418 

dysenteriae,  419 

meleagridis,  416 
Anaerobic,  47 

spore-producing  group,  189,  340 
Anaphylactin,  177 
Anaphylaxis,  128,  176 

active,  178 

bacterial,  178 

in  tuberculosis,  322 

passive,  178 

specificity,  178 
Anaplasma,  456,  465 

marginale,  465 
Anaplasmosis,  465 
Anilin  water,  103 

Animal  inoculation,  animals  used,  118 
for  isolation  of  pure  cultures,  109 
methods,  118 
reasons  for,  117 

kingdom,  divisions  of,  26 
Anopheles,  464 
Anthrax,  331 

group,  188,  331 
Anti-abrin,  145 
Anti-amboceptors,  159 
Anti-anaphylaxis,  178 
Antibacterial  immunity,  128 
Antibiosis,  56 

Antibodies  as  factors  in  acquired  im- 
munity, 127 

definition,  126,  127 

in  anaphylaxis,  177 
Anticomplement,  160 
Antienzymes,  145 
Antifonnin,  255,  312 
Antigens,  definition,  127 
Anfigonococcus  serum,  226 
Antipepsin,  145 
AntiphthiHn,  319 
Antirennet,  145 
Antiricin,  1  l.~» 
Antiseptic  surgery,  191 


Antiseptics,  52 
Antistreptococcic  sera,  203 
Antitoxic  immunity,  128 
Antitoxins    as    factors  in  immunity. 
127 

constitution  of,  134 

definition,  131 

diagrammatic  representation,  135 

diphtheria,  concentration,  142 
manufacture,  136 

Ehrlich's  theory  of  production,  133 

of  commercial  importance,  136 

tetanus,  preparation,  143 

standardization,  144 
Antivenoms,  145 
Aphthous  fever,  479 
Apiosoma  bigeminum,  456 
Apoplectiform  septicemia  of  fowls,  210 
Archispores,  455 
Argas  miniatus,  450 

persicus,  448,  450 

reflexus,  450 

Arnold  steam  sterilizer,  84,  85 
Arthritis,  202 
Arthrospores,  35,  37 
Ascomycetes,  40,  43 
Ascospores,  395 

of  molds,  42,  43 

of  Peziza,  43 

of  yeasts,  40 
ASCIIS,  42,  395 

of  Peziza,  43 
Aseptic  surgery,  192 
Asiatic  cholera,  369 

group,  189 
Aspergillosis,  395 
Asprrgillus,  393 

flavus,  398 

fumigatus,  395 

glaums,  394,  400 

niger,  399 

nigrcHcens,  400 

spore  production,  43 

subfuscus,  400 

toxin  production,  130 
Attenuation  of  bacteria,  124,  173 
methods  of,  124 

Autoclave,   S5,  86 

Autocyto  toxins,  164 


1XDKX 


499 


Autogenic  vaccine,  173 

from  M.  aureus,  195 
Autolysins,  1<>2 
Autolytic  enzymes,  60,  61 
A  v< 'lines  of  infection,  185 
Avian  diphtheria,  348 
Azotobacter,  70,  71 

agilis,  70 

chroococcum,  70 

BABESIA,  456 

bigeminum  bovis,  456 

parva,  458 

Babesiosis,  bovine,  456,  45^ 
Bacillary  dysentery,  2>2 
Bacillus  abortus,  341 
aceti,  63 

aerogenes  capsulatus,  361 
anaerobicus  cryptobutyricus,  361 
anthracis,  331 

symptomatic!,  355 
avisepticus,  294,  295 
of  Babes,  260 
botulinus,  349,  364 

production  of  toxins,  131 
bovicida,  302 
bovisepticus,  294,  302 
bulgaricus,  63 
butyricus,  64 
rudaveris  butyricus,  361 
capsulatus  mucosus,  267 
carrier,  281,  282 
chauveaui,  355 
chauvei,  349,  355 
cholera?,  295 

gallinarum,  29.~) 

suis,  262,  271,  481 
da  vat  us,  236 
coli  communis,  262 

isolation  from  water,  288 

reduction  of  nitrates,  66 
cuniculicida,  294,  303 
of  Danysz,  262,  276 
denitrificans,  67 
diphtheria?,  231 

production  of  toxins,  131 

toxin,  236 

vitulorum,  345 

Wesbrook's  types,  233 


Bacillus  dysenteric,  262,  278,  2s2 

types  of,  283 

< -mphysematis  vagina?,  361 
emulsion  of  Koch,  320 
enteritidis,  262,  268 

production  of  toxins,  131 

sporogenes,  361 

type  A,  275 

type  B,  275 
equisepticus,  294,  303 
erysipelatis  suis,  244 
feseri,  355 
filiformis,  345 
of  Flexner,  282 
fa?calis  alkaligenes,  262,  278 
of  Gartner,  268 
gastromycosis  ovis,  349,  359 
genus,  77,  78 
of  Hoffmann,  236 
of  Johnes'  disease,  308,  326 
of  Kutscher,  260 
lacti  morbi,  331,  338 
lactici  acidi,  204 
lactis  aerogenes,  262 
lepne,  308,  328 

lymphangitidis  ulcerosa,  238,  241 
mallei,  250 

murisepticus,  244,  248 
neapolitanus,  262 
necrophorus  group,  345 
necrosus,  345 
of  Xicolaier,  349 
cedematis,  359 

maligni,  359 
paratyphosus,  262,  274 
pastorianum,  70 
perfringens,  36] 
pestis,  295-303 

bubonica?,  303 
of  Pfeiffer,  239 

phlegmones  emphysematosa?,  361 
pleurisepticus,  293 
pneumonia?,  262,  267 
of  Preisz,  238 
prodigiosus  pigment,  56 
pseudodiphthericus,  231,  236 
pseudofarcy,  241 
pseudotuberculosis,  238 

group,  238 


500 


INDEX 


Bacillus   pseudotuberculosis   niurium, 
238 

ovis,  238 

psittacosis,  262,  276 
pullorum,  262,  277 
pyelonephritidis  bovis,  242 
pyocyaneus,  228 

pigment  production,  56 

toxins,  131 
pyogenes,  266 

bovis,  236,  242 

foetidus,  262 

suis,  228,  230 
radicicola,  71 
renalis  bovis,  242 
rhusiopathiae  suis,  244 
Salmoni,  271 
of  Selter,  260 

septicaemiae  haemorrhagicae,  293 
shape,  27 
of  Shiga,  282 
of  tetanus,  :^49 
smegmatis,  329,  330 
subtilis  group,  331,  333 
suicida,  298 
suipestifer,  271,  481 
suisepticus,  294,  298 
tetani,  349 

production  of  toxins,  131 
tuberculosis,  308 
typhi,  278 

abdominalis,  278 

murium,  262,  276 
typhosus,  262,  278 
welchii,  349,  361 
Hacteremia,  187 
Bacteria,  acetic  acid,  64 
amphitrichous,  34 
and  disease,  116 
arthrospores,  35 
atiiehous,  34  • 
Brownian  movement,  35 
butyric  acid,  64 
capsule,  31 
cell  inclusions,  34 
coil-wall,  31 
chromogenic,  56 
chromoparous.  66 
dec*:  .  M 


Hacteria,  distribution,  61 

ectoplast,  32 

endospores,  :>."> 

enzymes,  58 

filamentous,  27 

flagella,  34 

food  preservation,  74 

glycogen,  34 

grouping  of  cells,  'Js 

groups  of,  pathogenic,  1ST,  188 

histology  of,  31 

in  retting,  74 

in  sauerkraut,  74 

in  silage,  74 

in  tanning,  74 

in  tobacco  curing,  74 

involution  forms,  '27 

iron,  68 

lactic  acid,  63 

legume,  71 

lophotrichous,  35 

measuring,  101 

metachromatic  granules,  34 

monotrichous,  34 

nitrate,  69 

nitrite,  69 

nitrogen  fixing,  70 

normal  to  body,  184 

nucleus,  33 

of  the  colon,  184 

of  the  genito-urinary  organs,  185 

of  the  intestines,  1  s  1 
^of  the  mouth,  1S4 
*of  the  skin,  1S4 

of  the  stomach,  184 

of  water,  284 

oil  globules,  34 

oxidizing,  67 

peritrichous,  :;."> 

polar  staining,  34 

protoplasm,  32 

putrefactive,  64,  <>.""> 

reducing,  66 

reproduction,  ;}"> 

rapidity.  :;:, 

shape.  L'7 
sli. 'allied,  32 

size  of,  :;<» 

spores,  ii") 


IXDKX 


501 


Bacteria,  structure  of,  31 

sulphur,  67 

vacuoles,  34 
Barteriacea?,  77 
Bactericidal  action  of  sera,  157 
Ba«-t  erins,  1"3 

for  M.  aureus,  195 
Bacteriology,  definition,  17 

medical,  18 

scope  of,  17 

veterinary,  18 
Barteriolysins,  157,  I'-N 

normal,  160,  161 
Bacteriolytic  immunity,  128 

sera,  161 

Bacteriopurpurin,  46,  49 
Bacterium  abortum,  341 

aerogenes,  266 

anthracis,  331 

avicidum,  295 

hipolare  multicidum,  302 

coli  commune,  262 

diphtheria?,  231 

genus,  78 

lepra-.  328 

mallei,  250 

phosphoreum,  57 

tuberculosis,  308 

welchii,  361 

Bail's  aggressin  hypothesis,  181 
Balantidium  coli,  474 
Balbiana,  456 
Baleri,  433 

Bang's  abortion  bacillus,  341 
Barber  pipette,  107 
Basidiomycetes,  40 
Bed-bug,  445 
Beef  broth,  91 
Beerwort,  92 
Beggiatoa  sp.  67 
Biliaryr  fever  of  dog,  461 
Biochemical  tests,  96 
Bismarck  brown,  103 
Black  death,  305 
Blackhead  of  turkeys,  470 
Blackleg,  355 


coccidioides.  3S4.  389 
dermatitidis,  384,  386 


Blast  omyces  farciminosus,  384 

group,  189,  383 
Blastomycetes,  383 

ascospores,  40 

cell  inclusions,  38,  39 

chlamydospores,  40 

classification  of,  82 

cytoplasm,  39 

form,  38 

glycogen,  39 

grouping,  38 

morphology,  37 

nucleus,  39 

oil  globules,  39 

protoplasm,  38,  39 

size,  38 

spores,  40 

vacuoles,  39 
Blastomycotic  dermatitis,  386 

epizootic  lymphangitis,  384 
Blepharoplast,  424 
Blood  serum  as  a  medium,  94 
cultures,  111 

medium  for  B.  tuberculosis,  312 
recognition  of,  by  precipitins,  155 
stain,  106 

Blue-green  alga,  26 
Boophilus  bovis,  457 

decoloratus,  467 

Bordet-Gengou  phenomenon,   163 
Botryomycomata,  227 
Botryomycosis,  226 
Botryococcus  ascoformans,  226 
Botulism,  364 
Bouillon,  91 
Bovine  farcy,  378 
Bradsot,  359 
Braxy,  359 
Broth,  beef,  91 
extract,  91 

glycerin,  92 

serum,  92 

sugar,  92 
free,  91 

Brownian  movement,  35 
Bryophytes,  26 
Buboes,  305 
Biiffelseuche,  303 
Butter  bacillus,  329 


502 


INDEX 


CACHEXIAL  fever,  438 
Calf  diarrhea,  265,  266 
Calmette's  ophthalmo-reaction,  323 
Capsule,  31 

bacterial,  composition  of,  31 
Capybara,  432 

Carbolic  acid  as  disinfectant,  53 
Caseous  lymphangitis,  bovine,  238 
Castor  oil  bean  as  a  source  of  toxin, 

130 

Catalyst,  59 
Cattle  plague,  480 
Cell  inclusions  in  bacteria,  34 
glycogen,  34 

metachromatic  granules,  34 
of  molds,  40 
of  protozoa,  44 
of  yeasts,  38 
oil  globules,  34 
polar  granules,  34 
receptors,  132 
vacuoles,  34 
wall  of  bacteria,  31 
Cellulitis,  suppurative,  202 
Cellulose,  25 
Centrosome,  424 
Charbon,  331 

symptomatique,  355 
Chemicals,  effect  on  microorganisms, 

50 

Chemotaxy,  50 
Chemotropism,  52 
negative,  50 
positive,  50 
Chicken  cholera,  295 

pest,  486 

Chief  agglutinins,  151 
Chitin,  25,  31 

chemical  composition  of,  31 
Chlamydobacteriacese,  77,  80 
Chlamydospores  of    chlamydomucor, 

43 

of  molds,  42,  43 
of  yeasts,  40 
Cholera  bacillus,  295 
nostras,  371 
spirillum  group,  367 
Chromogenic  bacteria,  56 
Chromoparous  bacteria,  56 


Chronic  enteritis  of  cattle,  326 
i  Cilia,  44,  413 
Ciliophora,  414 
Cladothrix  actinomyces,  375 

genus,  77,  80,  372 
Classification,  history,  2C 

Migula's,  76 

of  animal  kingdom,  26 

of  bacteria,  76 

of  microorganisms,  75 

of  molds,  82 

of  plant  kingdom,  26 

of  protozoa,  412 

of  thallophytes,  26 

yeasts,  82 
Clostridium,  36 

pastorianum,  70 
Coagglutinin,  150 
CoccaceaD,  77 

Coccidioidal  granuloma,  389 
Coccidiosis  of  cattle,  473 

of  fowls,  470 

of  rabbit,  472 

of  sheep,  473 
Coccidium,  456,  469 

cuniculi,  472 

tenellum,  469 
Coccus,  27 
Colon  bacillus,  262 

subgroup,  261,  262 
Colon-typhoid  group,  261 
Columella,  43 
Comma  bacillus,  369 
Commensals,  46,  56 
Complement,  158 

absorption  of,  163 

action  of,  158 

fixation  of,  163 

specificity,  158 

structure  of,  163 
Complementoid,  159 
Complementophilous  haptophore,  159 
Conidia,  37,  42 

of  Aspornillus,  43 

of  Penicillium,  43 
Conidiophores,  42,  394 

of  Aspergillus,  43 

of  Penicillium,  43 
Conjunctiva!  tuberculin  reaction,  323 


1M)KX 


503 


Conorhinus  sp.,  437 
Consumption,  316 
Contact  beds,  292 
Contagious  abortion  in  cow,  341 
in  mares,  213 

diseases,  183 

of  cattle,  480 
Copula,  158,  464 
Corynebacterium  diphtheriae,  231 

pseudodiphthericum,  236 
Cowpox,  488 

Cresols  as  disinfectants,  53 
Crithridia,  438 

Cryptococcus  farciminosus,  3S4 
Culex,  465 
Cultural  characters  of  organisms,  110 

media,  preparation,  89 
Cutaneous  tuberculin  reaction,  323 
Cyanophycese,  26 
Cytase,  158 
Cytolysins,  157 

group,  160 

Cytophilous  haptophore,  159 
Cytorhyctes  vaccinia?,  488 
Cytotoxins,  157,  165 

DECAY,  64 
Delhi  boil,  439 
Deneke's  spirillum,  371 
Denitrification,  66 
Deodorant,  52 

Desiccation,  resistance  to,  by  micro- 
organisms, 46 
Deuxieme  vaccin,  336 
Diastase,  59 
Differentiation  of  plants  and  animals, 

25 
Diphtheria,  231 

antitoxin  concentration,  142 
manufacture,  136 
standardization,  138 

avian,  348 

group,  188,  231 

toxin,  manufacture,  136 

standardization,  137,  140 
Diplococcus,  28,  78 

gonorrho2a3,  224 

intracellularis  equi,  220 
meningitidis,  218 


Diplococcus  lanceolatus,  215 

of  Neisser,  224 

pneumonia?,  215 
Discomyces  bovis,  375 
Disease,  pathogenic  theory  of,  17 
Disinfectants,  52 

carbolic  acid,  53 

efficiencyj  determination,  99 

formaldehyd,  54 

lime,  53 

mercuric  chlorid,  53 

phenol,  53 

salts,  heavy  metals,  53 

sulphurous  acid,  53 
Distemper,  canine,  485 

in  equines,  207 
Distribution  of  bacteria,  61 
Dog  distemper,  485 
Dosing  chamber,  291 
Double  serum,  247 
Dourine,  426 
Drug  habituation,  130 
Dum-dum  fever,  438 
Dung  bacillus,  329 
Dunham's  solution,  92 

use  in  indol  test,  98 
Duration  of  immunity,  126 
Dysentery,  amebic,  419 

paratubercular,  of  cattle,  326 

EAST  African  coast  fever,  458 

tick  fever,  447 
Eberth  bacillus,  278 
Eberth-Gaffky  bacillus,  278 
Eclampsia,  puerperal,  179 
Ectoplast,  bacterial,  32 
mold,  42 
yeast,  39 
Egg  as  a  medium,  95 

medium   for  Bacillus  tuberculosis, 

312 
Ehrlich's  humoral  theory,  126 

lateral-chain  theory  of    immunity, 

131 

theory  of  cell  nutrition,  131,  132 
!  Electricity,  effect  of,  50 
Elements  necessary  for  cells,  45 
Encystment,  413 
I  Endocarditis,  ulcerative,  201 


504 


INDEX 


Endogenous  infection,  186 
Endospores,  35 

germination,  36 
Ent  amoeba,  416 

africana,  422 

coli,  416,  418 

histolytica,  416,  419 

nipponica,  416 

tetragena,  416,  422 
Enteral  introduction  of  organisms, 
Enteritidis  subgroup,  261,  268 
Enteritis,  chronic,  of  cattle,  326 
Enterohepatitis,  470 
Enzymes,  58 

autolytic,  60,  61 

diastase,  59 

extracellular,  58,  61 

gelatinase,  59 

hydrolytic,  59 

intracellular,  58,  61 

invertase,  59 

oxidases,  59 

oxidizing,  59 

pepsin,  59 

ptyalin,  59 

reducing,  60 

rennet,  59 

splitting,  59 

zymase,  58,  59 

Kpithelioma  contagiosum,  487 
Epizootic  lymphangitis,  404 
lit  I  nine  biliary  fever,  458 
Erysipelas,  201 

swine,  244 
Eubacteria,  77 
Kurythermic  bacteria,  48 
Exanthema,  187 
I  !\OK<  -nous  infection,  185 
External  resistance  of  body,  121 


FACULTATIVE  bacteria,  47 

Farcin  du  bceuf,  378 

Farcy,  250 

Favus,  409 

I  <  rrnentation,  58 

acetic  acid,  64 

acid,  62 

alcoholic,  62 


Fermentation,  butyric-  acid,  l>4 

lactic  acid,  63 

tubes,  96 
Ferments,  acetic  acid,  64 

acid,  62 

alcoholic,  62 

butyric  acid,  64 

lactic  acid,  63 

organized,  58 
176       unorganized,  58 
Filterable  virus,  476 
Filtration,  sterilization  by,  87 
Fixateur,  158 
Fixation  of  complement,  164 

in  diagnosis  of  glanders,  256 
Fixed  virus,  491 
Flagella  of  bacteria,  34 

of  protozoa,  44,  413 

stain,  van  Ermengem's,  105 

Loffler's,  105 
Fluorescin,  229 
Fomite,  183 
Food  relationships  o'  microorganisms, 

45 

Foot-and-mouth  disease,  479 
Foot  rot  of  cattle,  348 

of  sheep,  202 
Formaldehyd,  54 
Fowl  cholera,  295 

diphtheria  group,  188 

plague,  486 

pox,  487 

septicemia,  367 

Friedlander's  pneumococous,  2(i7 
Fuchsin,  carbol-,  103 

phenol,  103 

Ziehl's,  103 
Fungi,  26 

imprrfecti,  392 
Fusarium,  393,  400,  402 

corallinum,  402  ' 


f  1  ALL  sickness,  435,   !'>"> 
( iallionclla  forrutfinra,  t'>x 
Galziekte,  435,  It,:, 
Gambian  horse  sickness,  432 
'  ..is  production,  tests  for,  96 
(  lasmus  cdcnia,  301 


INDEX 


(  ia.-ometer.  97 
Gelatin  nutrient,  93 
plate  culture,  112 
stab  cultures,  111 
( ielatinase.  .")«.» 
Gentian  violet,  anilin,  103 

aqueous,  103 
( lennicide,  52 
Cibberetta.  402 
Glanders,  250 

group,  188,  250 
( ilossinia  morsitans,  431 
palpalis,  431,  435,  437 
( Ilycerin  agar,  93 
broth,  92 
gelatin,  93 

Glycogen  in  bacteria,  34 
Coat  pox,  488 
Gonococcus.  224 
Gonorrhea.  224 
Grain's  stain,  106 
Granular  vaginitis  in  cattle,  211 
Granulobacillus  saccharobutyricus  im- 

mobilis,  361 

Gra<s  bacillus  of  Moeller,  329 
Groups  of  pathogenic  organisms,  187 
abortion  bacillus,  189 
acid-fast,  188 
actinomyces,  189 
anaerobic  spore-producing,  189 
anthrax,  188 
blastomyces,  189 
diphtheria,  188 
fowl  diphtheria,  188 
glanders,  188 

hemorrhagic  septicemia,  188 
hyphomycetes,  189 
intestinal,  188 
necrosis  bacillus,  189 
non-specific    pyogenic    bacilli, 

188 

coccus,  188 

pseudotuberculosis,   188 
specific  coccus,  188 
spirillum,  189 
spirochete,  189 
swine  erysipelas,  188 
Growth  temperature  range,  48 
Gruber-Widal  test,  microscopic,  151 


HSMATOCOCCUS,  456 

bo  vis,  45«l 

Haffkine's  vaccine.  306 
Haltcridiuni,  456,  465 
Hanging  drop,  101 
Haptophore,   complementophilous.  of 

amboceptor,  159 
cytophilous,  of  amboceptor,  15!» 
of  agglutinin,  148 
of  antitoxin,  134 
of  enzyme,  145 
of  precipitin,  154 
of  toxin,  134 
Hautschicht,  39 
Healing  by  first  intention,  191 
Heine-Medin  disease,  488 
Helcosoma  tropicum,  439 
Heliotropism,  52 
Hemagglutinms,  153 
Hemoglobinuria  in  sheep,  460 
Hemolysins,  157,  162 
Hemoproteus,  456,  465 
Hemorrhagic  septicemia  group,    188 

293 

of  birds,  294,  295 
of  cattle,  294,  302 
of  horses,  294,  303 
of  man,  295,  303 
of  rabbits,  294,  303 
of  rodents,  295,  303 
of  swine,  294,  302 
Hemotoxin,  131 

of  Bacillus  pyocyaneus,  230 
Hepatization  of  lungs,  216 
Hepatozoon  perniciosuni,  467 
Herpes  tonsurans,  408 
Herpetomonas,  423,  4:>s 
donovani,  438 
infantum,  439 
Hctcrologons,  148 
,  Heterolysins.  162 
Hilfkorper,   15s 
Histology  of  bacteria,  31 
of  blast omycetes,  37 
of  hyphomycetes,  41 
of  molds,  41 
of  saccharomycetes,  37 
of  yeasts.  37.  38 
Hog  cholera,  271.  4M 


500 


INDEX 


Hog-cholera  subgroup,  261,  268 
Homologous,  148 
Horse  pox,  488 

sickness,  483 

syphilis,  426 
Hot-air  oven,  84 
Humors' of  body  in  Ehrlich's  theory, 

126 

Hundstaupe,  485 
Hydrochaerus  capybara,  432 
Hydrophobia,  489 
Hydrotropism,  52 
Hyperimmunization,  161 
Hypersusceptibility,  176 
Hypha,  41,  392 
Hyphomycetes,  40,  392 

form,  40 

group,  189,  392 

histology,  41 

morphology,  40 

reproduction,  42 

size,  40 
Hypobosca  rufipes,  436 

IMMUNE  body,  158 

in  anaphylaxis,  177 
Immunity,  acquired,  122,  123 

active,  122,  123 

definition,  121 

duration,  127 

history  of,  23 

individual,  122 

natural,  122 

passive,  122 

racial,  122 

specific,  122 

theories  of,  125 

to  anaphylaxis,  178 

unit  of  diphtheria  antitoxin,  139 

of  tetanus  antitoxin,  144 
Immunkorper,  158 
Indol,  composition,  66 

test  for,  98 
Infantile  kala-azar,  439 

paralysis,  488 
Infection  atrium,  185 

endogenous,  186 

exogenous,  185 

mixed,  187 


Infection,  non-specific,  186 

phlogistic,  187 

primary,  187 

secondary,  187 

specific,  186 
Infectious  anemia,  484 

disease,  183 
Inflammation,  190 
Infusoria,  414,  474 
Ingestion,  inoculation  by,  119 
Inhalation,  inoculation  by,  119 
Intermediate  subgroup,  261,  268 
Intermittent  sterilization,  84,  85 
Internal  resistance  of  body,  121 
Intestinal  group,  188,  361 
Intracardiac  injection,  119 
Intracranial  injections,  118 
Intradermal  tuberculin  reaction,  323 
Intraocular  injections,  119 
Intraperitoneal  injection,  methods,  118 
Intrathoracic  injection,  119 
Intravenous  inoculation  of  rabbit,  US 
Invertase,  59 

Involution  forms  of  bacteria,  27 
Iron  bacteria,  68 
Isolysins,  162 
Itch  disease,  402 
Ixidoplasma  bigeminum,  456 

JEQUIRITY  bean  as  source  of  toxin,  130 

KALA-AZAR,  438 
infantile,  439 
Kinetonucleus,  424 
Klebs-Loffler  bacillus,  231 
Koch's  rules,  116 
difficulties,  116 
statement,  116 

Konew's  precipitation  test   for  glan- 
ders, 255 

Lo  dose  diphtheria  toxin,  141 
L+  dose  diphtheria  toxin,  141 
Laboratory  methods,  history,  22 
Lactic-acid  fermentation,  ('>:; 
Legume  bacteria,  71 
Irishman-Donovan  bodies,  438 
L< 'ish mania  donovani,  438 
farciminosa,  384 


IXDKX 


507 


Leishmania  infantum,  439 

tropica,  439 
Leprosy,  32s 
Leptothrix  genus,  77,  80,  372 

ochracea,  68 
Leukocidin,  194 

of  Bacillus  pyocyaneus,  230 
Leukocytozoon,  456,  467 
Light,  disinfecting  action  of  rays,  49 

relationships  of  microorganisms,  49 
Lip  and  leg  ulceration  of  sheep,  348 
Lockjaw,  349 
Louse,  body,  446 
Lumbar  puncture,  220 
Lumpy  jaw,  375 
Lung  plague,  478 
Lupus,  316 
Lymphangitis,  epizootic,  384,  404 

ulcerative,  241 
Lyssa,  489 

MACROGAMETES,  469 
Macronucleus,  44 
Macrophages,  166 
Madura  foot,  381,  382 
Mai  de  caderas,  431 
Maladie  du  coit,  426 
Malaria,  463 

Malarial  catarrhal  fever,  460 
Malignant  carbuncle,  331 

edema,  359 

jaundice  of  dog,  461 
Malignes  oedem,  359 
Mallease,  256 
Mallein,  258 
Malleinum  siccum,  259 
Malta  fever,  221 
Mammitis,  cattle,  214 
Marginal  points,  465 
Mastigophora,  414,  415,  423 
Mastitis,  202 

cattle,  214 

Maul-und-Klauenseuche,  479 
Maximum  growth  temperature,  48 
Meat  differentiation  by  precipitation 
test,  155 

poisoning,  364 

due  to  Bacillus  botulinus,  364 
due  to  Bacillus  entoritidis,  268 


Media,  beef -broth,  91 

from  beef  extract,  91 

beerwort,  92 

blood-serum,  94 
Loffler's,  95 

bouillon,  91 

Dunham's  solution,  92 

egg,  95 

glycerin  broth,  92 

milk,  92 

nutrient  agar,  93 
gelatin,  93 

potato,  94 

serum  broth,  92 

sugar  broth,  92 

sugar-free  broth,  92 

synthetic,  92 

Uschinsky's  solution,  92 
Medical  bacteriology,  18 
Mediterranean  fever,  221 
Meningitis,  218 

human  epidemic,  218 
Meningococcus,  218 
Mercuric  chlorid,  53 
Merismopedia,  78 
Merozoites,  469 
Mesophilic  bacteria,  48 
Metachromatic  granules,  34 
Metastatic  infections,  119 
Metatrophic  bacteria,  46 
Metazoa,  26,  43,  412 
Methods,  staining,  102 
Methylene-blue,  Gabbett's,  103 

Loffler's,  102 

Micrococcus  albus,  191,  196 
toxins,  131 

ascoformans,  207,  226 

aureus,  191,  192 
toxins,  131 

botryogenus,  226 

bovis,  191,  196 

caprinus,  207,  222 

cereus  albus,  191,  197 
flavus,  191,  197 

citreus,  191,  196 

epidermidis  albus,  191,  197 

gonorrhoea?,  207,  224 

intrarellularis  equi,  207,  220 

lanceolatus,  207,  215 


508 


INDEX 


Micrococcus  mastitidis,  191,  196 

melitonsis.  'J07,  221 

meningitidis,  207,  218 

ovis,  191,  197 

pneumoniae,  207,  215 

pyogenes  alb  us,  196 
aureus,  192 
bo  vis,  196 
citreus,  196 

weichselbaumii,  218 
!\ I  irrogametocytes,  469 
Micron,  30,  101 
Micronucleus  of  protozoa,  44 

of  trypanosomes,  424 
.Microphages,  166 
Microscope  and  its  influence,  18 
Microspira,  79 

comma,  369 

metschnikovi,  367 
Microsporon,  393,  404,  408 

adouini,  408 

carinum,  408 

equinum,  408 

furfur,  408 
Milk  as  a  medium,  92 

cultures  in,  111 

litmus,  92 

cultures  in,  112 

sickness,  338 
Milzbrand,  331 
Minimum  lethal  dose  of  diphtheria 

toxin,  137 
of  tetanus  toxin,  144 

temperature,  48 
Mist  bacillus,  329 

Moisture  relationships  of  microorgan- 
isms, 46 

Mold  group,  392 
Molds,  classification,  82 

form,  40 

histology,  41 

morphology,  40 

nitrogen  fixing,  71,  72 

reproduction,  42 

size,  40 

structure,  41 
Monilia  Candida.,  410 
Mordant,  102 
Morphology  of  bacteria,  2."..    :i 


Morphology  of  blast  omycetes,  37 

of  hyphomycetes,  40 

of  molds,  40 

of  saccharomycetes,  37 

of  yeasts,  37 
Morve,  250 
Morvin,  258 
Motor  nucleus,  424 
Mouse  septicemia,  248 
Mucin,  32 
Mucor,  42,  43 

sporangium,  43 

zygospore,  43 
Mud  fever,  484 
Mycelium,  41,  392 
Mycetoma,  381,  382 
Mycobacterium  diphtheria?,  231 

leprae,  328 

mallei,  250 

pseudodiphthericum,  236 

pseudotuberculosis,  238 

tuberculosis,  308 
Mycorrhiza,  72 
Myxosporidia,  455 

NAGANA,  429 

Natural  immunity,  122 

Navel-ill,  191 

Necamspora,  402 

Necrosis  bacillus  group,  189,  345 

Necrotic  dermatitis,  347 

metritis,  348 

pox,  347 

scratches,  347 

vaginitis,  348 

vulvitis,  348 
Negative  phase,  172 
Negri  bodies.  4s(.» 
Nephelometer,  174 
Nephrolysins,  157 
Nessler's  solution,  96 
Neurocytes  hydrophobise,  489 
Xcurotoxin,   131 
Neu  tuberculin  of  Koch,  320 
Nitrate  bacteria,  69 
Nitrification,  69 
Nitrites,  tests  for,  98 
Nitrogen  cycle,  73 

fixation,  70 


IXDKX 


Nitrogen,  non-symbiotic,  70 

symbiotic.  71 
Nitroso-bacteria,  69 
Nitrosomonas  europea,  69 

javensis,  69 
Xocardia  t'arcinic'a,  378 

genus,  77,  80,  372 
Xodules  in  tuberculosis,  316 

on  roots  of  legumes,  71 
Non-specific  pyogenic  bacillus  group, 

iss.  22s 

coccus  group,  188,  190 
Normal  solutions,  89 
Noxious  retention  theory  of  immunity, 

126 
Nucleus  molds,  41 

protozoa.  44 

yeasts.  :>'.» 

Nutrient  broth,  cultures  in,  111 
Nutrients  required  by  bacteria,  90 


malin,  359 
Oidium,  393,  408 

albicans,  410 

coccidioides,  389 

dermatitidis,  386 

porrigenis,  409 

type  of  spore,  42,  43 
(  )il  globules,  34 

immersion,  objective,  100 

optics  of,  100 
Omphalophlebitis,  201 
or.cyte,  469 
Ookinete,  464 
Oospora,  393,  408 

porrigenis,  409 

Ophthalmo-tubereulin  reaction,  323 
(  )pilacao,  437 
Opsonic  index,  169 

materials  needed,  169,  170 
McCampbell's  modification,   171 
method,  171 
Opsonins,  128,  166 

immune,  168 

normal,  168 

occurrence,  167 

Optimum  growth  temperature,  48 
Organella,  44,  412 
Oriental  sore,  439 


Ornithodorus  moubata,  446,  448 
Osmotic  pressure,  adjustment  of  or- 
ganisms to,  55 

Ovine  caseous  lymphadenitis  2:;^ 
Ovopla.sm  nrit-ntale,  439 
Oxidase,  59 
Oxidation  by  bacteria  of  ammonia,  tin 

of  hydrogen  sulphid,  <>7 

of  iron,  68 

of  nitrites,  69 
Oxy tuberculin,  319 

PARACOLON  bacillus,  274 
Parat'ormaldehyd,  54 
Paraphyses,  43 
Parasites,  46 
Paratrophic  bacteria,  46 
Paratubercular    dysentery    of    cattle, 

326 

Paratyphoid,  274 

Parenteral  injection  of  organisms,  176 
Passive  anaphylaxis,  178 

immunity,  122,  124 
Pasteurella,  293 
Pasteurellosis,  293 

of  birds,  294,  295 

of  cattle,  294,  302 

of  horses,  294,  303 

of  man,  295,  303 

of  rabbits,  294,  303 

of  rodents,  295,  303 

of  swine,  294,  298 
Pasteur  method  of  vaccination  against 

hydrophobia,  490 

Pasteurization   of  milk  and  Strepto- 
coccus lacticus,  205 
Pathogenic  organism,  definition,   119 
Pearl  disease,  316 
Pediculus  vestimenti,  446 
Penicillium,  393,  399 

glaucum,  401 

spore  production,  43 
Pepsin,  59 
Peptonization,  65 
Period  of  incubation,  130 
Peripneumonia,  478 
Perithechim,  395 
J  Peritonitis,  201 
Perlsuche,  316 


510 


INDEX 


Pernicious  anemia,  484 

Petri  dish,  108 

Peziza,  43 

Pfeiffer's  phenomenon,  157 

Pferdepest,  483 

Phagocytes,  classification,  166 

definitions,  126,  166 
Phagocytosis,  theory  of,  126, 166 
Phenol  as  disinfectant,  53 
Phenomenon  of  Arthus,  177 

of  Theobald  Smith,  187 
Philocytase,  158 
Phlogistic  disease,  187 
Photogenic  bacteria,  57 
Phycomycetes,  40 
Physiological  characters,  115 

salt  solution,  56 

Physiology  of  microorganisms,  45 
antibiosis,  46 
antiseptic,  52 
chemicals,  50 
commensalism,  46 
disinfectants,  52 
electricity,  50 
fermentations,  58 
food  relationships,  45 
light,  49 

production,  57 
moisture  relationships,  46 
pigment  production,  46 
respiration,  47 
symbiosis,  46 
temperature,  48 
Pigments,  bacterial,  56 

diffuse,  56 
Piroplasma,  456 
bigeminum,  456 
canis,  461 
commune,  462 
equi,  458 
gibsoni,  462 
unit-ana,  458 
ovi-s  460 
parvum,  458 

Piroplasmosis,  bovine,  456,  458 
canine,  461,  462 
equine,  4> 
ovine,  460 
Planococcus  genus,  77 


Planosarcina,  77 

Plant  kingdom,  divisions  of,  26 

Plasmodium,  456,  462 

falciparum,  465 

immaculatum,  465 

malarise,  464 

vivax,  463 
Plasmolysis,  33,  55 
Plasmoptysis,  33 
Plate  cultures,  108 
Pleuropneumonia,  478 

epizootic,  of  equines,  214 
Pneumobacillus,  215,  267 
Pneumococcus,  215 
Pneumomycosis,  395 
Pneumonia,  215 

traumatic,  201 
Polar  staining,  34 
Poliomyelitis,  488 
Pollan  in,  145 

Pollen  as  source  of  toxin,  130 
Polyvalent  vaccines,  173 
Positive  phase,  172 
Potato  as  a  medium,  94 

cultures,  110 
Pox,  488 
Precipitation,  128,  147 

bacterial  differentiation,  156 

blood  stain,  differentiation,  155 

group,  154 

meat  differentiation,  155 

uses,  154 

Precipitin,  147,  154 
Precipitogen,  154 
Precipitoid,  154 

Pn -disposing  factors  to  disease,  122 
Premier  vaccine,  336 
Preparator,  158 

Presumptive  test  for  Bacillus  coli,  288 
Proteins,  decomposition,  65 
Proteolysis,  65 
Proteosoma,  456,  It.:. 
Prototrophic  bacteria,  -Hi 
Protozoa,  form  and  size.  1  \ 

histology,  44 

morphology,  43 

pathogenic,  412 

reproduction,  44 
Pseudofarcy,  241,  ::M 


INDEX 


511 


Pseudoglanders,  241 
Pseudomonas  aeruginosa,  228 
genus,  78 
pyocyanea,  228 
Pseudopodia,  413 

of  protozoa,  44,  413 
Pseudotuberculosis  group,  188,  238 
Psittacosis,  276 
Psychrophilic  bacteria,  48 
Pteridophytes,  26 
Ptomains,  65 
Ptyalin,  59 
Puerperal  fever,  201 
Pure  culture  methods,  animal  inocu- 
lation, 118 
dilution,  107 
direct  isolation,  107 
plating,  108 
smearing,  107 
use  of  differential  antiseptics, 

109 

of  heat,  109 
Pus,  191 
Putrefaction,  64 
Pyelonephritis,  242 
Pyemia,  187 

produced  by  Streptococcus  pyog- 

enes,  201 
Pyocyanin,  229 
Pyogenic  organisms,  190 
Pyrosoma,  456 

bigeminum,  456 
Pythogenic  theory  of  disease,  23 

QUARTAN  malaria,  464 
Quarter  evil,  355 
ill,  355 

RABIES,  489 
Rauschbrand,  355 
Reactivation,  157 
Receptors,  cell,  132 
Recurrent  fever,  444,  446 
Red  fever  of  swine,  244 
Reducing  enzymes,  60 
Reduction  of  chlorates,  67 

of  nitrates,  66 

of  sulphates,  66 

processes,  66 


Reduction,  tests  for,  97,  98 
Relapsing  fever,  444,  446 
Rennet,  59 
Reproduction  hi  bacteria,  35 

in  hyphomycetes,  41 

in  molds,  41 

rapidity,  35 

Respiration  of  microorganisms,  defini- 
tion, 47 

Rheumatism,  202 
Rhipicephalus  annulatus,  4")  7 

appendiculatus,  458 

bursa,  460 

decoloratus,  450 

simus,  458 
Rhisopoda,  414,  415 
Rhodesian  red-water,  458 

tick  fever,  458 
Rice-water  stools,  Asiatic  cholera,  369, 

371 

Ricin,  130 

Ricinus  communis  as  source  of  toxin, 
130 

zanzibarensis    as    source    of    toxin 

(ricin),  130 
Rigor  mortis,  60 
Rinderpest,  480 
Rinderseuche,  302 
Ring  test  for  glanders,  255 
Ringworm,  408 
Rotlauf ,  244 

doppelserum,  247 
Rotz,  250 
Rouget,  244 
Roup,  470 

SACCHAROMYCES,  383 

cerevisiae,  62,  82 

dermatitidis,  386 

genus,  82 
Sarrharomycetes  ascosporus,  40 

cell  inclusions,  38,  39 

chlamydospores,  40 

classification,  82 

cytoplasm,  39 

ectoplast,  39 

form,  38 

glycogen,  39 

grouping,  38 


512 


INDEX 


Saccharomycetes,  morphology,  37 

nucleus,  39 

oil  globules,  39 

protoplasm,  38 

reproduction,  38 

size,  38 

spores,  40 

vacuoles,  39 
Salmonella,  271 

Salts  of  the  heavy  metals  as  disin- 
fectants, 53 
Sand  filtration,  292 
Sanitary  science,  23 
Sapremia,  187 
Saprophytes,  46    • 
Saprozoites,  46 
Sarcina  genus,  77 

grouping,  29 
Sarcocystis,  456,  468 

bertrami,  467 

Lendemanni,  469 

miescherinia,  469 

muris,  468 

tenella,  469 
Sarcodina,  414,  415 
Sarcoptes  equi,  404 
Sarcoptic  dermatitis,  402 
Sarcosporidiosis,  468 
Sausage  poisoning,  364 
Scarification  as  means  of  inoculation, 

119 

Schizomycetes,  26 
Schizonts,  469 
Schizophycete,  26 
Schweinepest,  481 
Schweineseuche,  298 
Sclerotium,  392 
Scrofula,  316 
Sedimentation,  290 
Srn-ibilisin,  177 
Sensibilisinogen,  177 
Sensitization,  128 
Srptir  tank,  291 
Septicemia,  187 

of  fowls,  367 

gangreneuse,  3-V.i 

produced    by  Streptocorm<    pyog- 

enes,  201 
S.-ptiridin,  298 


Septum,  41 

Serum  antidiphtheriticum,  138 

broth,  «.»•_> 

sickness,  176 
Sewage  disposal,  291 
Sheaths,  bacterial,  32 
Sheep  box,  488 
Side-chain,  definition,  132 
Skatol,  66 

Sleeping  sickness,  436 
Smallpox,  488 
Sorehead,  487 
Souma,  434 
Soumaya,  434 

Sources  of  food  for  microorganisms,  45 
Specific  coccus  group,  188,  207 

infection,  186 
Sperm  atophyt  es,  26 
Spirillaceae,  77 
Spirillosis  of  cattle,  450 

of  fowls,  448 

of  horses,  450 

of  man,  444,  446 

of  sheep,  450 
Spirillum  anserirui,  448 

cholera,  367,  369 
asiaticac,  369 
isolation  from  water,  288 

of  Deneke,  371 

desulfuricans,  67 
.   duttoni,  446 

of  Finkler  and  Prior,  371 

genus,  77,  79 

group,  189,  367 

marchouxi,  448 

metschnikovi,  Mt>7 

nicollei,  4  Is 

obermeieri,  444 

ovina,  450 

pallidum,  451 

phosphorescent,  M71 

shape,  27 

theilni,    }:.<> 

tyrogenum,  371 
Spirorli;i'ta  arixTma,  44o,  44s 

dcnticola,  441 

dcntiiim,   HI 

duttoni,  443,  U». 

etjui,  450 


IXDEX 


513 


Spirochaeta  evansi,  428 

gallinarum,  443,  448 

genus,  77,  SO,  423,  439,  443 

inequalis,  441 

kochi,  447 

novyi,  448 

obermeieri,  443,  444 

ovina,  444 

ovis,  443 

pallida,  451 

perfringens,  443 

pertenuis,  454 

pinnae,  440 

recta,  441 

tenuis,  441 

theileri,  450 

undulata,  441 
Spirochete  group,  189,  440 
Spirochetosis  of  fowls,  448 
Spirophyllum  ferrugineum,  68 
Spiroschaudinnia,  443 

duttoni,  446 

recurrentis,  444 
Spirosoma,  79 
Splenic  fever,  331 
Splitting  enzymes,  59 
Spontaneous  generation,  20 
Sporangiophore,  42 

of  mucor,  43 

of  sporodinia,  43 
Sporangium  of  mucor,  43 

of  sporodinia,  43 
Spore  case,  42 
Spores,  bacterial,  35 
germination,  36 

of  hyphomycetes,  42 

of  molds,  42 

of  mucor,  43 

of  penicillium,  43 

of  peziza,  43 

of  sporodinia,  43 

of  yeast,  40 

staining  of,  104 
Sporoblasts,  4.V, 
Sporodinia,  43 
Sporotrichosis,  404 
SporotrirhiiMi.  385,  393,  404 

adouini,  408 

Beurmanni,  404 


Sporotrichum  schenkii,  404 

tonsurans,  408 
Sporozoa,  414,  455 
Sporozoites,  455,  469 
Stable  pneumonia,  214 
Staining  methods,  acid-fast,  104 
acid  alcohol,  104 
Gabbett's  method,  104 
blood,  106 
flagella,  105 
Loffler's,  105 
van  Ermengem,  105 
for  Bacillus  tuberculosis,  104 
Giemsa's,  106 
Gram's,  106 
preparation  of  mount,  103 

protozoan,  106  , ^ 

Romanowsky,  106 
spore  stain,  104 
Hansen,  104 
Moller's,  104 
Stains,  acid,  102 
basic,  102 

Bismarck  brown,  103 
carbol  fuchsin,  103 
Gabbett's,  103 

Loffler's  methylene-blue,  102 
mordants,  102 
phenol  fuchsin,  103 
Stalactite  formation  of  Bacillus  pestis, 

304 

Staphylococcus,  29 
aureus,  192 
citreus,  196 
pyogenes  albus,  196 
aureus,  192 
bo  vis,  196 
citreus,  196 
Staphylolysin,  194 
Starters,  206 
Stegomyia,  488 
Stenothermic  bacteria,  48 
Sterigma,  394 

Sterigmata  of  aspergillus,  43 
Sterigmatocystis,  393,  394 
Sterilization,  83 

:it  low  temperatures,  87 
by  chemicals,  87 
by  filtration,  87 


514 


INDEX 


Sterilization  by  flame,  87 

by  hot  air,  83 

by  steam  under  pressure,  85 

by  streaming  steam,  84 

intermittent,  84,  85 
Sterilizer,  Arnold  steam,  84,  85 
Steinberg  bulb,  99 
Stomoxys  calcitrans,  428 
Strangles,  207 

Strauss'  biologic  test  for  glanders,  254 
Street  virus,  491 
Streptobacillus,  29 

Streptococcus   agalactiae   contagiosae, 
214 

articulorum,  197 

capsulatus  gallinarum,  210 

contagious  abortion  in  mares,  213 

coryzae  contagiosae  equorum,  207 

equi,  207 

erysipelatos,  197 

gallinarum,  207,  210 

genus,  28,  77 

lacticus,  63,  191,  204 

mastitidis  sporadic*,  214 

meningitidis,  218 

Ostertag,  211 

pneumoniae,  215 

puerperalis,  197 

pyogenes,  191,  197 
malignis,  197 

scarlatinosus,  197 

septicus,  197 
Streptolysin,  200,  202 
Streptothricosis  in  cattle,  375 

in  dogs,  380 

in  goats,  330 

in  man,  381 
Streptothrix,  81,  372 

actinomyces,  375 
'  bovis,  375 

canis,  380 

caprae,  380 

coelicolor,  374 

cuniculi,  345 

eppingeri,  382 

farcinica,  378 

freeri,  382 

madurse,  381 

necrophora,  345 


Streptothrix  nocardii,  378 
Structure  of  protozoa,  412 
Subcutaneous  inoculation  methods, 

118 

Subdural  inoculation,  119 
Substance  sensibilisatrice,  158 
Sulphur  dioxid  as  disinfectant,  53 
Sulphurous  acid  as  disinfectant,  53 
Suppuration,  190 
Surra,  428 
Susceptibility,  121 

variations  in,  122 
Swamp  fever,  484 
Swine  erysipelas,  244 
group,  188,  244 

fever,  481 

plague,  298 

pox,  488 

Symbion,  symbiont,  56 
Symbiosis,  56 
Symptomatic  anthrax,  355 
Synthetic  action  of  microorganisms, 
61 

media,  92 
Syphilis,  451 

TABANUS  fumifer,  429 
Takosis,  222 

Temperature  relationships  of  micro- 
organisms, 48 
Tertian  malaria,  463 
Tetanolysin,  353 
Tetanospasmin,  353 
Tetanus,  349. 

antitoxin,  preparation  of,  144 

standardization  of,  144 
toxin,  preparation  of,  144 

standardization  of,  144 
Tetracoccus,  28 
Texas  fever,  456 
Thallophytrs,  '2(\ 

classification  of,  26 
Thrili-riii  parva,  45S 
Theories  of  immunity,  125 

Khrlirh's  humoral,  126 

exhaustion,   125 

Metchnikoff's,  126 

noxious  n-lrnt ion,  12G 
Thrrmal  death-point,  48 


INDEX 


515 


Thermal  death-point,  conditions  gov- 
erning, 48,  49 

method  of  determination,  99 
Thermophilic  bacteria,  48 
Thiobacteriacejr,  77 
Thiophysa  volutans,  67 
Thiothrix,  77,  81 
Thrush,  410 
Tick  fever,  446 
in  cattle,  456 
in  sheep,  460 
Rhodesian,  458 
Tinea  favosa,  409 
Torula,  82,  383,  384 
Toxemia,  187 
Toxin,  tetanus,  preparation  of,  143 

standardization  of,  144 
Toxine,  129 
Toxins  as  factors  in  immunity,  127 

characteristics  of,  129 

constitution,  134 

definition,  129 

diagrammatic  representation,  135 

manufacture,  136 

of  pollen,  130 

preferential  union  with  body  cells, 
135 

sources,  130 

specificity,  131 
Toxoid,  134 
Toxone,  142 
Toxophore  of  toxin,  134 
Trembles,  338 
Treponema,  443 

pallidum,  451 

pertenuis,  454 
Trichomycetes,  372 
Trichophyton,  393,  404,  408 

megalosporon,  408 

microsporon,  408 

tonsurans,  408 
Trickling  filter,  292 
Tropisms,  51 
Trypanosoma,  423 

brucei,  429 

calmettei,  437 

castellani,  436 

cazalboui,  434 

congolense,  433 


Trypanosoma  cruzi,  437 
dimorphon,  432 
donovani,  438 
elmassiani,  431 
equinum,  431 
equiperdum,  426 
evansi,  428 
gambiense,  436 
lewisi,  437 
pecaudi,  433 
rougeti,  420 
theileri,  435 
ugandense,  436 
vespertilionis,  425 
Trypanosomes,  423 
cultivation,  426 
morphology,  424 
of  birds,  437 
Tse-tse  fly,  429,  431,  437 
Tubercle,  miliary,  316 
Tuberculin,  317 
Alt's,  318,  321 
Denys',  319 
Hirschf elder's,  319 
Klebs',  319 
Koch's  old,  318,  321 

purified,  319 
Landmann's,  319 
new,  of  Koch,  320 
reaction    as  anaphylactic  reaction, 

180 

T.  O.,  319 
T.  R.,  319 

tests,  anaphylactic,  nature  of,  322 
conjunctival,  323 
cutaneous,  323 
Calmette's,  323 
intradermal,  323 
ophthalmo-,  323 
subcutaneous  injection,  321 
von  Pirquet's,  323 
Wolf-Eisner,  323 
Tuberculinum  O.,  319 

R.,  319 

Tuberculocidin,  319 
Tuberculol,  319 
Tuberculosis,  308 
Turgor,  .V> 
Types  of  immunity,  122 


510 


IXDEX 


Typhoid  dysentery  subgroup,  261,  278 
fever,  278 

ULCERATIVE  lymphangitis,  241 
litramicroscope,  19,  476 
Ultramicroscopic  organisms,  476 

virus,  31 

Undulating  membrane,  424 
Univalent  vaccines,  173 
Uschinsky's  solution,  92 

VACCINATION,  definition,  125,  161 

to  increase  opsonins,  172 
Vaccines,  autogenic,  173 

bacterins,  173 

polyvalent,  173 

preparation,  173 

univalent,  173 
Vacuoles,  34 

in  bacteria,  34 
Vaginitis,     contagious     granular,     of 

cattle,  211 
Vegetative  rod,  36 
Verrucose  vaginitis  of  cattle,  211 
Vibrio,  79 

cholerae,  369 

group,  367 

metschnikovi,  367 

proteus,  371 
Vibrion  septique,  359 
Virulence,  definition,  119 
Virus,  IV! 

filterable,  31 

ultramicroscopic,  31 
von  Pirquet's  cutaneous  reaction,  323 

.KM ANN  syphilis  test,  164 
Water,  bacteria  of,  284 

bacterial  standards,  _'^7 

purification,  284,  2 

qualitative  examination,  iN7 

quantitative  examination,  285 
interpretation,  liMl 

-ell-purification,  2M» 
\\  eiuerf-  hypothesis.    189 
We*  African  tick  fever,  446 


White  scours  in  calves,  265,  266 
diarrhea,  470 

of  chicks  caused  by  Bacillus  pul- 

loruin,  277 

by  Coccidium  tenollum,  469 
Widal  test,  151 

macroscopic  153,  281 
microscopic,  151,  152,  281 
Wildseuche,  302 

Wolf-Eisner  tuberculin   reaction,  323 
Wooden  tongue,  375 
Woolsorter's  disease,  331 
Wround  infection,    Streptococcus   py- 
ogenes,  200 

YAWS,  454 

Yeast  ascospores,  40 

cellulose,  38 

chlamydospores,  40 

classification,  82 

spores,  40 
Yeasts,  cell  inclusions,  38,  39 

cytoplasm,  39 

ectoplast,  39 

form,  38 

glycogen,  39 

grouping,  38 

morphology,  37      , 

nucleus,  39 

oil  globules,  39 

protoplasm,  38 

reproduction,  38 

size,  38 

vacuoles,  39 
Yellow  fever,  488 

ZOOGLEA,  30 

pulmonis  equi,  226 
Zwischenkorper,  158 
Zygospore,  42,  43 

of  mucor,  43 
Zymase,  58,  59 
Zymophore  of  agglutinins,  148 

of  complement,   lf>(.) 

of  enzymes,  145 

of  precipitins,  !."»:; 
Zymotoxic  group  of  agglutinins,  149 


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Keen's  Surgery,  Kelly  and  Noble's  Gynecology  and  Abdominal  Sur- 
gery, Cabot's  Differential  Diagnosis,  Mumford's  Surgery,  Cotton's  Dis- 
locations and  Joint  Fractures,  Crandon's  Surgical  After-treatment, 
Sisson's  Veterinary  Anatomy,  Anders  and  Boston's  Medical  Diagnosis, 
Bonney's  Tuberculosis,  Gant's  Constipation  and  Obstruction,  Jordan's 
Bacteriology,  and  Kemp  on  Stomach  and  Intestines.  These  books 
have  made  for  themselves  a  place  among  the  best  works  on  their 
respective  subjects. 

A    Complete    Catalogue    of  our  Publications   will    be  Sent  upon    Request 


SAUNDERS'    BOOKS   ON 


Jordan's 
General    Bacteriology 

A  Text-Book  of  General  Bacteriology.  By  EDWIN  O.  JORDAN,  PH.D., 
Professor  of  Bacteriology  in  the  University  of  Chicago  and  in  Rush 
Medical  College.  Octavo  of  594  pages,  illustrated.  Cloth,  $3.00  net. 

THE  NEW  (2d)  EDITION 

Professor  Jordan's  work  embraces  the  entire  field  of  bacteriology,  the  non- 
pathogenic  as  well  as  the  pathogenic  bacteria  being  considered,  giving  greater 
emphasis,  of  course,  to  the  latter.  There  are  extensive  chapters  on  methods  of 
studying  bacteria,  including  staining,  biochemical  tests,  cultures,  etc. ;  on  the 
development  and  composition  of  bacteria  ;  on  enzymes  and  fermentation-products; 
on  the  bacterial  production  of  pigment,  acid  and  alkali  ;  and  on  ptomains  and 
toxins.  Especially  complete  is  the  presentation  of  the  serum  treatment  of  gonor- 
rhea, diphtheria,  dysentery,  and  tetanus.  The  relation  of  bovine  to  human 
tuberculosis  and  the  ocular  tuberculin  reaction  receive  extensive  consideration. 

This  work  will  also  appeal  to  academic  and  scientific  students.  It  contains 
chapters  on  the  bacteriology  of  plants,  milk  and  milk-products,  air,  agriculture, 
water,  food  preservatives,  the  processes  of  leather  tanning,  tobacco  curing,  and 
vinegar  making  ;  the  relation  of  bacteriology  to  household  administration  and  to 
sanitary  engineering,  etc. 

Prof.  Severance  Burrage,  Associate  Professor  of  Sanitary  Science,  Purdue  University. 

"  I  am  much  impressed  with  the  completeness  and  accuracy  of  the  book.  It  certainly 
covers  the  ground  more  completely  than  any  other  American  book  .that  I  have  seen." 


Buchanan's 
Veterinary   Bacteriology 

Veterinary  Bacteriology.  By  ROBERT  E.  BUCHANAN,  Ph.D.,  Pro- 
fessor of  Bacteriology  in  the  Iowa  State  College  of  Agriculture  and 
Mechanic  Arts.  Octavo  of  500  pages,  with  214  illustrations. 

JUST  READY 

Professor  Buchanan's  new  work  is  a  comprehensive  one,  presenting  the  prac- 
tical side  of  bacteriology  as  applied  in  veterinary  science,  discussing  thoroughly 
all  bacteria  causing  diseases  of  the  domestic  animals.  The  author  has  gone  mi- 
nutely into  the  consideration  of  immunity,  opsonic  index,  reproduction,  sterili/.a- 
tion,  antiseptics,  biochemic  tests,  cultu»e  media,  isolation  of  cultures,  the  manu- 
facture of  the  various  toxins,  antitoxins,  tuberculins,  and  vaccines  that  have 
proved  of  diagnostic  or  therapeutic  value.  Then,  in  addition  to  bacteria  and 
protozoa  proper,  he  considers  molds,  mildews,  smuts,  rusts,  toadstools,  puff-balls, 
and  the  other  fungi  pathogenic  for  animals.  Professor  Buchanan  is  a  forceful 
writer,  and  his  book  has  all  the  earmarks  of  a  master  of  his  subject. 


HISTOLOGY  AND  PHYSIOLOGY. 


Dtirck  and  Hektoen's 

General  Pathologic  Histology 

Atlas  and   Epitome  of   General  Pathologic  Histology.      By   PR. 

DR.  H.  DURCK,  of  Munich.  Edited,  with  additions,  by  LUDVIG  HEK- 
TOEN,  M.  D.,  Professor  of  Pathology  in  Rush  Medical  College,  Chicago. 
172  colored  figures  on  77  lithographic  plates,  36  text-cuts,  many  in 
colors,  and  353  pages.  Cloth,  $5 .00  net.  In  Saunders"  Hand- Atlas  Series. 


This  new  Atlas  will  be  found  even  more  valuable  than  the  two  preceding 
volumes  on  Special  Pathologic  Histology,  to  which,  in  a  manner,  it  is  a  com- 
panion work.  The  text  gives  the  generally  accepted  views  in  regard  to  the  signifi- 
cance of  pathologic  processes,  explained  in  clear  and  easily  understood  language. 
The  lithographs  in  some  cases  required  as  many  as  twenty-six  colors  to  reproduce 
the  original  painting.  Dr.  Hektoen  has  made  many  additions  of  great  value. 

W.  T.  Councilman,  M.  D., 

Professor  of  Pathologic  Anatomy,  Harvard  University. 

"  I  have  seen  no  plates  which  impress  me  as  so  truly  representing  histologic  appearances 
as  do  these.     The  book  is  a  valuable  one." 

Howell's  Physiology 


A  Text-Book  of  Physiology.  By  WILLIAM  H.  HOWELL,  PH.D., 
M.  D.,  Professor  of  Physiology  in  the  Johns  Hopkins  University,  Balti- 
more, Md.  Octavo  of  999  pages,  296  illustrations.  Cloth,  $4.00  net. 

THE  NEW   (3d)  EDITION 

Dr.  Howell  has  had  many  years  of  experience  as  a  teacher  of  physiology  in 
several  of  the  leading  medical  schools,  and  is  therefore  exceedingly  well  fitted  to 
write  a  text-book  on  this  subject.  Main  emphasis  has  been  laid  upon  those  facts 
and  views  which  will  be  directly  helpful  in  the  practical  branches  of  medicine.  At 
the  same  time,  however,  sufficient  consideration  has  been  given  to  the  experimen- 
tal side  of  the  science.  The  entire  literature  of  physiology  has  been  thoroughly 
digested  by  Dr.  Howell:  and  the  important  views  and  conclusions  introduced  into 
his  work.  Illustrations  have  been  most  freely  used. 

The  Lancet.  London 

"  This  is  one  of  the  best  recent  text-books  on  physiology,  and  we  warmly  commend  it  to  the 
attention  of  students  who  desire  to  obtain  by  reading  a  general,  all-round,  yet  concise  survey  of 
the  scope,  facts,  theories,  and  speculations  that  make  up  its  subject  matter." 


SAUNDERS*  BOOKS  ON 


McFarland's   Pathology 


A  Text-Book  of  Pathology.  By  JOSEPH  McFARLAND,  M.  D.,  Pro- 
fessor of  Pathology  and  Bacteriology  in  the  Medico-Chirurgical  College 
of  Philadelphia.  Octavo  of  856  pages,  with  437  illustrations,  many  in 
colors.  Cloth,  $5.00  net;  Half  Morocco,  $6.50  net. 

THE   NEW    (2d)    EDITION 

You  cannot  successfully  treat  disease  unless  you  have  a  practical,  clinical 
knowledge  of  the  pathologic  changes  produced  by  disease.  For  this  purpose  Dr. 
McFarland's  work  is  well  fitted.  It  was  written  with  just  such  an  end  in  view — to 
furnish  a  ready  means  of  acquiring  a  thorough  training  in  the  subject,  a  training 
such  as  would  be  of  daily  help  in  your  practice.  For  this  edition  every  page  has 
been  gone  over  most  carefully,  correcting,  omitting  the  obsolete,  and  adding  the 
new.  Some  sections  have  been  entirely  rewritten.  You  will  find  it  a  book  well 
worth  consulting,  for  it  is  the  work  of  an  authority. 

St.  Paul  Medical  Journal 

"  It  is  safe  to  say  that  there  are  few  who  are  better  qualified  to  give  a  re'sume'  of  the  modern 
views  on  this  subject  than  McFarland.  The  subject-matter  is  thoroughly  up  to  date." 

Boston  Medical  and  Surgical  Journal 

"  It  contains  a  great  mass  of  well-classified  facts.  One  of  the  best  sections  is  that  on  the 
special  pathology  of  the  blood." 


McFarland's 

Biology:  Medical  and  General 

Biology:  Medical  and  General — By  JOSEPH  McFARLAND,  M.  D., 
Professor  of  Pathology  and  Bacteriology  in  the  Medico-Chirurgical  Col- 
lege of  Phila.  I2mo,  440  pages,  160  illustrations.  Cloth,  $1.75  net . 

ILLUSTRATED 

This  work  is  both  a  general  and  medical  biology.  The  former  because  it  dis- 
cusses the  peculiar  nature  and  reactions  of  living  substance  generally;  the  latter 
because  particular  emphasis  is  laid  <>n  those  subjects  of  special  interest  and  value 
in  the  study  and  practice  of  medicine.  The  illustrations  will  be  found  of  great 
assistance. 

Frederic  P.  Gorh&m,  A.  M.,  Drown  University. 

"  I  am  greatly  pleased  with  it.  Perhaps  the  highest  praise  which  I  can  give  the  book  is  to 
say  that  it  more  nearly  approaches  the  course  I  am  now  giving  in  general  biology  than  any 
other  work." 


BA CTERIOLOG  Y  AND  HISTOLOG  Y. 


McFarland's 
Pathogenic  Bacteria 

The  New  (6th)  Edition,  Revised 


A  Text-Book  Upon  the  Pathogenic  Bacteria.  By  JOSEPH  McFAR- 
LAND,  M.  D.,  Professor  of  Pathology  and  Bacteriology  in  the  Medico- 
Chirurgical  College  of  Philadelphia,  Pathologist  to  the  Medico-Chirur- 
gical  Hospital,  Philadelphia,  etc.  Octavo  volume  of  709  pages,  finely 
illustrated.  Cloth,  $3.50  net. 

FULLY  ILLUSTRATED 

This  book  gives  a  concise  account  of  the  technical  procedures  necessary  in  the 
study  of  bacteriology,  a  brief  description  of  the  life-history  of  the  important  patho- 
genic bacteria,  and  sufficient  description  of  the  pathologic  lesions  accompanying 
the  micro-organismal  invasions  to  give  zm  idea  of  the  origin  of  symptoms  and  the 
causes  of  death.  The  illustrations  are  mainly  reproductions  of  the  best  the  world 
affords,  and  are  beautifully  executed.  In  this  edition  the  entire  work  has  been 
practically  rewritten,  old  matter  eliminated,  and  much  new  matter  inserted. 

H .  B.  Anderson,  M.  D., 

Professor  of  Pathology  and  Bacteriology,  Trinity  Medical  College,  Toronto. 
"  The  book  is  a  satisfactory  one,  and  I  shall  take  pleasure  in  recommending  it  to  the  students 
of  Trinity  College." 

The  Lancet,  London 

"  It  is  excellently  adapted  for  the  medical  students  and  practitioners  for  whom  it  is  avowedly 
written.  .  .  .  The  descriptions  given  are  accurate  and  readable." 

Hill's   Histology  and  Org'anography 

A  Manual  of  Histology  and  Organography.  By  CHARLES  HILL, 
M.  D.,  formerly  Assistant  Professor  of  Histology  and  Embryology, 
Northwestern  University,  Chicago.  I2mo  of  468  pages,  337  illustra- 
tions. Flexible  leather,  $2.00  net. 

THE  NEW  (2d)  EDITION 

Dr.  Hill's  work  is  characterized  by  a  completeness  of  discussion  rarely  met  in 
a  book  of  this  size.  Particular  consideration  is  given  the  mouth  and  teeth. 

Pennsylvania  Medical  Journal 

"  It  is  arranged  in  such  a  manner  as  to  be  easy  of  access  and  comprehension.  To  any 
contemplating  the  study  of  histology  and  organography  we  would  commend  this  work." 


SAUNDERS    BOOKS   ON 


GET  A  •  THE  NEW 

THE  BEST  /\  m  C 1?  1 C  Sill  STANDARD 

Illustrated   Dictionary 

Just  Ready— New  (6th)  Edition,  Entirely  Reset— A  New  Work 


The  American  Illustrated  Medical  Dictionary.  A  new  and  com- 
plete dictionary  of  the  terms  used  in  Medicine,  Surgery,  Dentistry, 
Pharmacy,  Chemistry,  Veterinary  Science,  Nursing,  and  kindred 
branches ;  with  over  100  new  and  elaborate  tables  and  many  handsome 
illustrations.  By  W.  A.  NEWMAN  BORLAND,  M.D.,  Editor  of  "  The 
American  Pocket  Medical  Dictionary."  Large  octavo,  935  pages, 
bound  in  full  flexible  leather.  Price,  $4.50  net ;  with  thumb  index, 
$5.00  net. 

IT  DEFINES  ALL  THE  NEW  WORDS— IT  IS  UP  TO  DATE 

Borland's  Dictionary  defines  hundreds  of  the  newest  terms  not  defined  in  any 
other  dictionary — bar  none.  These  new  terms  are  live,  active  words,  taken 
right  from  modern  medical  literature. 

It  gives  the  capitalization  and  pronunciation  of  all  words.  It  makes  a  feature  of 
the  derivation  or  etymology  of  the  words.  In  some  dictionaries  the  etymology 
occupies  only  a  secondary  place,  in  many  cases  no  derivation  being  given  at  all. 
In  "  Dorland,"  practically  every  word  is  given  its  derivation. 

In  "Dorland"   every  word  has  a  separate  paragraph,  thus  making  it  easy  to 

find  a  word  quickly. 

The  tables  of  arteries,   muscles,    nerves,    veins   etc.,    are    of  the   greatest   help 

in  assembling  anatomic  facts.      In  them  are  classified  for  quick  study  all  the 

necessary  information  about  the  various  structures. 

In    "Dorland"    every   word    is    given    its    definition — a    definition   that    defines 

in  the  fewest  possible  words.      In  some  dictionaries  hundreds  of  words  are  not 

defined  at  all,  referring  the  reader  to  some  other  source  for  the  information  he 

wants  at  once. 

Howard  A.  Kelly,  M.  D.,  Johns  Hopkins  University,  Baltimore 

"  Dr.  Dorland's  dictionary  is  admirable.  It  is  so  well  gotten  up  and  of  such  convenient 
size.  No  errors  have  been  found  in  my  use  of  it." 

J.  Collins  Warren,  M.  D.,  LL.D..  F.R.C.S.  (Hon.).  Harvard  Medical  School 

"  I  regard  it  as  a  valuable  aid  to  my  medical  literary  work.  It  is  very  complete  and  of 
convenient  site  to  handle  comfortably.  I  use  it  in  preference  to  any  other." 


PATHOLOGY. 


Stengel's 
Text-Book  of  Pathology 


The    New  (5th)   Edition 


A  Text-Book  of  Pathology.  By  ALFRED  STENGEL,  M.  D.,  Professor 
of  Medicine  in  the  University  of  Pennsylvania.  Octavo  volume  of  979 
pages,  with  400  text-illustrations,  many  in  colors,  and  7  full-page 
colored  plates.  Cloth,  $5.00  net;  Sheep  or  Half  Morocco,  $6.50  net. 

WITH  400  TEXT-CUTS.  MANY  IN  COLORS.  AND  7  COLORED  PLATES 

In  this  work  the  practical  application  of  pathologic  facts  to  clinical  medicine 
is  considered  more  fully  than  is  customary  in  works  on  pathology.  While  the 
subject  of  pathology  is  treated  in  the  broadest  way  consistent  with  the  size  of  the 
book,  an  effort  has  been  made  to  present  the  subject  from  the  point  of  view  of  the 
clinician.  In  the  second  part  of  the  work  the  pathology  of  individual  organs  and 
tissues  is  treated  systematically  and  quite  fully  under  subheadings  that  clearly 
indicate  the  subject-matter  to  be  found  on  each  page.  In  this  edition  the  section 
dealing  with  General  Pathology  has  been  most  extensively  revised,  several  of  the 
important  chapters  having  been  practically  rewritten.  A  very  useful  addition 
is  an  Appendix  treating  of  th'  technic  of  pathologic  methods,  giving  briefly  the 
most  important  methods  at  present  in  use  for  the  study  of  pathology,  including, 
however,  only  those  methods  capable  of  giving  satisfactory  results.  The  book 
will  be  found  to  maintain  fully  its  popularity. 


PERSONAL  AND  PRESS  OPINIONS 


William  H.  Welch.  M.  D.. 

Professor  of  Pathology,  Johns  Hopkins  University,  Baltimore,  Md. 

"  I  consider  the  work  abreast  of  modern  pathology,  and  useful  to  both  students  and  practi- 
tioners. It  presents  in  a  concise  and  well-considered  form  the  essential  facts  of  general  and 
special  pathologic  anatomy,  with  more  than  usual  emphasis  upon  pathologic  physiology." 

Ludvig  Hektoen,  M.  D.. 

Professor  of  Pathology,  Rush  Medical  College,  Chicago. 

"  I  regard  it  as  the  most  serviceable  text-book  for  students  on  this  subject  yet  written  by  an 
American  author." 

The  Lancet,  London 

"This  volume  is  intended  to  present  the  subject  of  pathology  in  as  practical  a  form  as  pos- 
sible, and  more  especially  from  the  point  of  view  of  the  'clinical  pathologist.'  These  subjects 
have  been  faithfully  carried  out,  and  a  valuable  text-book  is  the  result.  We  can  most  favorably 
recommend  it  to  our  readers  as  a  thoroughly  practical  work  on  clinical  pathology." 


SAUNDERS'    BOOKS   ON 


Mallory  and  Wright's 
Pathologic  Technique 

Just  Ready— New  (5th)  Edition,  Revised 


Pathologic  Technique.  A  Practical  Manual  for  Workers  in  Patho- 
logic Histology,  including  Directions  for  the  Performance  of  Autopsies 
and  for  Clinical  Diagnosis  by  Laboratory  Methods.  By  FRANK  B. 
MALLORY,  M.  D.,  Associate  Professor  of  Pathology,  Harvard  Univer- 
sity ;  and  JAMES  H.  WRIGHT,  M.  D.,  Director  of  the  Pathologic  Labora- 
tory, Massachusetts  General  Hospital.  Octavo  of  500  pages,  with  152 
illustrations.  Cloth,  $3.00  net. 

WITH  CHAPTERS  ON  POST-MORTEM  TECHNIQUE  AND  AUTOPSIES 

In  revising  the  book  for  the  new  edition  the  authors  have  kept  in  view  the 
needs  of  the  laboratory  worker,  whether  student,  practitioner,  or  pathologist,  for 
a  practical  manual  of  histologic  and  bacteriologic  methods  in  the  study  of  patho- 
logic material.  Many  parts  have  been  rewritten,  many  new  methods  have  been 
added,  and  the  number  of  illustrations  has  been  considerably  increased.  Among 
the  new  matter  are  the  following  :  Smith's  staining  method  for  encapsulated 
bacteria  ;  the  antiformin  method  for  detection  and  cultivation  of  tubercle  bacilli  ; 
Musgrave's  and  Clegg's  method  for  the  cultivation  of  amebae  ;  Wright's  method 
for  staining  myelin  sheaths  in  frozen  sections  ;  Ghoreyeb's  method  for  spirochetes  ; 
Alzheimer's  method  for  cytologic  examination  of  cerebrospinal  fluid  ;  Giemsa's 
new  method  for  protozoa  and  bacteria  in  sections,  and  the  Wassermann-Noguchi 
tests  for  syphilis. 


PERSONAL  AND  PRESS  OPINIONS 


Wm.  H.  Welch,  M.  D.. 

Professor  of  Pathology,  Johns  Hopkins  University,  Baltimore. 

"  I  have  been  looking  forward  to  the  publication  of  this  book,  and  I  am  glad  to  say  that  I 
find  it  a  most  useful  laboratory  and  post-mortem  guide,  full  of  practical  information  and  well 
up  to  date." 

Botton  Medical  and  Surgical  Journal 

"  This  manual,  since  its  first  appearance,  has  been  recognized  as  the  standard  guide  in  patho- 
logical technique,  and  has  become  well-nigh  indispensable  to  the  laboratory  worker." 

Journal  of  the  American  Medical  Association 

"  One  of  the  most  complete  works  on  the  subject,  and  one  which  should  be  in  the  library 
of  every  physician  who  hopes  to  keep  pace  with  the  great  advances  made  in  pathology." 


EMBRYOLOGY. 


Heisler's 
Text-Book  qf  Embryology 

Just  Ready— The  New  (4th)  Edition 


A  Text-Book  of  Embryology.  By  JOHN  C.  HEISLER,  M.D.,  Pro- 
fessor of  Anatomy  in  the  Medico-Chirurgical  College,  Philadelphia. 
Octavo  volume  of  450  pages,  with  212  illustrations,  32  of  them  in 
colors.  Cloth,  $3.00  net 

WITH    212     ILLUSTRATIONS.     32     IN     COLORS 

The  fact  of  embryology  having  acquired  in  recent  years  such  great  interest 
in  connection  with  the  teaching  and  with  the  proper  comprehension  of  human 
anatomy,  it  is  of  first  importance  to  the  student  of  medicine  that  a  concise  and 
yet  sufficiently  full  text-book  upon  the  subject  be  available.  This  new  edition 
represents  all  the  latest  advances  recently  made  in  the  science  of  embryology. 
Many  portions  have  been  entirely  rewritten,  and  a  great  deal  of  new  and  impor- 
tant matter  added.  A  number  of  new  illustrations  have  also  been  introduced  and 
these  will  prove  very  valuable.  The  previous  editions  of  this  work  filled  a  gap 
most  admirably,  and  this  new  edition  will  undoubtedly  maintain  the  reputation 
already  won.  Heisler's  Embryology  has  become  a  standard  work. 


PERSONAL  AND   PRESS  OPINIONS 


G.  Carl  Huber,  M.D., 

Professor  of  Embryology  at  the  IVistar  Institute,  University  of  Pennsylvania. 
"  I  find  the  second  edition  of  '  A  Text-Book  of  Embryology'  by  Dr.  Heisler  an  improve- 
ment on  the  first.     The  figures  added  increase  greatly  the  value  of  the  work.     I  am  again 
recommending  it  to  our  students." 

William  Wathen,  M.  D., 

Professor  of  Obstetrics,  Abdominal  Surgery,  and  Gynecology,  and  Dean,  Kentucky  School  of 

Medicine,  Louisville,  Ky. 

"  It  is  systematic,  scientific,  full  of  simplicity,  and  just  such  a  work  as  a  medical  student 
will  be  able  to  comprehend." 

Birmingham  Medical  Review,  England 

"  We  can  most  confidently  recommend  Dr.  Heisler's  book  to  the  student  of  biology  or 
medicine  for  his  careful  study,  if  his  aim  be  to  acquire  a  sound  and  practical  acquaintance  with 
the  subject  of  embryology." 


io  SAUNDERS'    BOOKS   ON 

Wells'  Chemical  Pathology 


Chemical  Pathology. — Being  a  Discussion  of  General  Pathology 
from  the  Standpoint  of  the  Chemical  Processes  Involved.  By  H. 
GIDEON  WELLS,  PH.  D.,  tyL  D.,  Assistant  Professor  of  Pathology  in  the 
University  of  Chicago.  Octavo  of  549  pages.  Cloth,  $3.25  net. 

A  PRACTICAL  BOOK 

Dr.  Wells'  work  is  written  for  the  physician,  for  those  engaged  in  research  in 
pathology  and  physiologic  chemistry,  and  for  the  medical  student.  In  the  intro- 
ductory chapter  are  discussed  the  chemistry  and  physics  of  the  animal  cell,  giving 
the  essential  facts  of  ionization,  diffusion,  osmotic  pressure,  etc.,  and  the  relation 
of  these  facts  to  cellular  activities.  Special  chapters  are  devoted  to  Diabetes  and 
to  Uric-acid  Metabolism  and  Gout. 

Win.   H.  Welch,  M.  D. 

Professor  of  Pathology,  Johns  Hopkins  University. 

"  The  work  fills  a  real  need  in  the  English  literature  of  a  very  important  subject,  and  I 
shall  be  glad  to  recommend  it  to  my  students." 

Lusk's 
Clements  of  Nutrition 

Elements  of  the  Science  of  Nutrition.  By  GRAHAM  LUSK,  PH.  D., 
Professor  of  Physiology  at  Cornell  Medical  School.  Octavo  volume 
of  302  pages.  Cloth,  £3.00  net. 

THE   NEW   (2d)    EDITION 

Prof.  Lusk  presents  the  scientific  foundations  upon  which  rests  our  knowledge 
of  nutrition  and  metabolism,  both  in  health  and  in  disease.  There  are  special 
chapters  on  the  metabolism  of  diabetes  and  fever,  and  on  purin  metabolism. 
The  work  will  also  prove  valuable  to  students  of  animal  dietetics  at  agricultural 
stations. 

L«wellyi  F.  Barker.  M.  D. 

Professor  of  the  Principles  and  Practice  of  Medicine,  Johns  Hopkins  University. 
"  1  shall  recommend  it  highly  to  my  students.     It  is  a  comfort  to  have  such  a  discussion 
of  the  subject  in  English." 


HISTOLOGY.  II 


Bohm,  Davidoff,  and 
Huber's  Histology 


A  Text-Book  of  Human  Histology.  Including  Microscopic  Tech- 
nic.  By  DR.  A.  A.  BOHM  and  DR.  M.  VON  DAVIDOFF,  of  Munich,  and 
G.  GARL  HUBER,  M.D.,  Professor  of  Embryology  at  the  Wistar  Insti- 
tute, University  of  Pennsylvania.  Handsome  octavo  of  528  pages,  with 
361  beautiful  original  illustrations.  Flexible  cloth,  $3.50  net. 

THE    NEW   (2d)    EDITION,    ENLARGED 

The  work  of  Drs.  Bohm  and  Davidoff  is  well  known  in  the  German  edition, 
and  has  been  considered  one  of  the  most  practically  useful  books  on  the  subject 
of  Human  Histology.  This  second  edition  has  been  in  great  part  rewritten  and 
very  much  enlarged  by  Dr.  Huber,  who  has  also  added  over  one  hundred  origi- 
nal illustrations.  Dr.  Huber's  extensive  additions  have  rendered  the  work  the 
most  complete  students'  text-book  on  Histology  in  existence. 

Boston  Medical  and  Surgical  Journal 

"  Is  unquestionably  a  text-book  of  the  first  rank,  having  been  carefully  written  by  thorough 
masters  of  the  subject,  and  in  certain  directions  it  is  much  superior  to  any  other  histological 
manual." 


DrewV 

Invertebrate  Zoology 

A  Laboratory  Manual   of    Invertebrate  Zoology.     By   OILMAN   A. 

DREW,  PH.D.,  Professor  of  Biology  at  the  University  of  Maine.  With  the 
aid  of  Members  of  the  Zoological  Staff  of  Instructors  of  the  Marine  Biolog- 
ical Laboratory,  Woods  Holl,  Mass.  i2mo  of  200  pages.  Cloth,  $1.25  net. 

A    LABORATORY    WORK 

The  subject  is  presented  in  a  logical  way,  and  the  type  method  of  study  has 
been  followed,  as  this  method  has  been  the  prevailing  one  for  many  years. 

Prof.  Allison  A.  Smyth,  Jr..  Virginia  Polytechnic  Institute 

"  I  think  it  is  the  best  laboratory  manual  of  zoology  I  have  yet  seen.     The  large  number 
of  forms  dealt  with  makes  the  work  applicable  to  almost  any  locality." 


12  SAUNDERS    BOOKS    ON 


Morris'   Cardiac   Pathology 

Studies  in  Cardiac  Pathology.  By  GEORGE  W.  NORRIS,  M.D., 
Associate  in  Medicine  at  the  University  of  Pennsylvania.  Large  octavo 
of  235  pages,  with  85  superb  illustrations.  Cloth,  $5.00  net. 

JUST  READY 

The  wide  interest  being  manifested  in  heart  lesions  makes  this  book  particu- 
larly opportune.  The  illustrations  are  superb  and  are  faithful  reproductions  of 
the  specimens  photographed.  Each  illustration  is  accompanied  by  a  detailed 
description ;  besides,  there  is  ample  letter  press  supplementing  the  pictures. 
Throughout  the  book  considerable  matter  of  a  diagnostic  and  therapeutic  nature 
has  been  interwoven,  making  the  work  more  valuable  to  the  general  physician. 
The  subjects  treated  are  endocarditis  ;  diseases  of  the  aortic,  mitral,  tricuspid,  and 
pulmonary  orifices  ;  pericarditis,  hypertrophy,  dilatation,  aneurism,  syphilis,  and 
congenital  lesions. 

McConnell's  Pathology 

A  Manual  of  Pathology.  By  GUTHRIE  McCoNNELL,M.D.,  Professor 
of  Bacteriology  and  Pathology  at  Temple  University,  Philadelphia, 
I2mo  of  523  pages,  with  170  illustrations.  Flexible  leather,  $2.50  net. 

JUST  READY— NEW  (2d)  EDITION 

Dr.  McConnell  has  discussed  his  subject  with  a  clearness  and  precision  of 
style  that  make  the  work  of  great  assistance  to  both  student  and  practitioner. 
The  illustrations  have  been  introduced  for  their  practical  value. 

New  York  State  Journal  of  Medicine 

"  The  book  treats  the  subject  of  pathology  with  a  thoroughness  lacking  in  many  works  of 
greater  pretension.  The  illustrations — many  of  them  original — are  profuse  and  of  exceptional 
excellence." 


Hektoen  and  Riesman's  Pathology 

AMERICAN  TEXT-BOOK  OF  PATHOLOGY.  Edited  by  LUDVIG  HEK- 
TOEN, M.D.,  Professor  of  Pathology,  Rush  Medical  College,  Chi- 
cago; and  DAVID  RIESMAN,  M.D.,  Professor  of  Clinical  Medicine, 
Philadelphia  Polyclinic.  Octavo  of  1245  pages,  443  illustra- 
tions, 66  in  colors.  Cloth,  $7.50  net ;  Half  Morocco,  $9.00  net. 


HISTOLOGY.  13 


Dtirck  and  Hektoen's 

Special    Pathologic    Histology 

Atlas  and  Epitome  of  Special  Pathologic  Histology.     By  DR.  H. 

DURCK,  of  Munich.  Edited,  with  additions,  by  LUDVIG  HEKTOEN,  M.  D., 
Professor  of  Pathology,  Rush  Medical  College,  Chicago.  In  two  parts. 
Part  I. — Circulatory,  Respiratory,  and  Gastro-intestinal  Tracts.  120 
colored  figures  on  62  plates,  and  158  pages  of  text.  Part  II. — Liver, 
Urinary  and  Sexual  Organs,  Nervous  System,  Skin,  Muscles,  and 
Bones.  123  colored  figures  on  60  plates,  and  192  pages  of  text.  Per 
part :  Cloth,  $3.00  net.  /;/  Sounders'  Hand- Atlas  Series. 

The  great  value  of  these  plates  is  that  they  represent  in  the  exact  colors  the  effect 
of  the  stains,  which  is  of  such  great  importance  for  the  differentiation  of  tissue. 
The  text  portion  of  the  book  is  admirable,  and,  while  brief,  it  is  entirely  satisfac- 
tory in  that  the  leading  facts  are  stated,  and  so  stated  that  the  reader  feels  he  has 
grasped  the  subject  extensively. 

William  H.  Welch,  M.  D., 

Professor  of  Pathology,  Johns  Hopkins  University,  Baltimore. 

"  I  consider  Diirck's  'Atlas  of  Special  Pathologic  Histology,"  edited  by  Hektoen,  a  very 
useful  book  for  students  and  others.  The  plates  are  admirable." 

Sobotta  and  Huber's 
Human  Histology 

Atlas  and  Epitome  of  Human  Histology.  By  PRIVATDOCENT  DR. 
J.  SOBOTTA,  of  Wiirzburg.  Edited,  with  additions,  by  G.  CARL  HUBER, 
M.  D.,  Professor  of  Histology  and  Embryology  in  the  University  of 
Michigan,  Ann  Arbor.  With  214  colored  figures  on  80  plates,  68 
text-illustrations,  and  248  pages  of  text.  Cloth,  $4.50  net.  In 
Sannders'  Hand- Atlas  Series. 

INCLUDING   MICROSCOPIC  ANATOMY 

The  work  combines  an  abundance  of  well-chosen  and  most  accurate  illustra- 
tions, with  a  concise  text,  and  in  such  a  manner  as  to  make  it  both  atlas  and  text- 
book. The  great  majority  of  the  illustrations  were  made  from  sections  prepared 
from  human  tissues,  and  always  from  fresh  and  in  every  respect  normal  specimens. 
The  colored  lithographic  plates  have  been  produced  with  the  aid  of  over  thirty  colors. 

Boston  Medical  and  Surgical  Journal 

"  In  color  and  proportion  they  are  characterized  by  gratifying  accuracy  and  lithographic 
beauty." 


14  SAUNDERS1    BOOKS    ON 

Bosanquet  on  Spirochaetes 

Spirochaetes :  A  Review  of  Recent  Work,  with  Some  Original  Ob- 
servations. By  W.  CECIL  BOSANQUET,  M.D.,  Fellow  of  the  Royal  Col- 
lege of  Physicians,  London.  Octavo  of  1 52  pages,  illustrated.  $2.50  net. 

JUST  READY 

This  is  a  complete  and  authoritative  monograph  on  the  Spirochaetes,  giving- 
morphology,  pathogenesis,  classification,  staining,  etc.  Pseudospirochastes  are 
also  considered,  and  the  entire  text  well  illustrated.  The  high  standing  of  Dr. 
Bosanquet  in  this  field  of  study  makes  this  new  work  particularly  valuable. 


Levy  and  Klemperer's 
Clinical  Bacteriology 

f  _^ — — — — — ^^^^— 

The  Elements  of  Clinical  Bacteriology.  By  DRS.  ERNST  LEVY  and 
FELIX  KLEMPERER,  of  the  University  of  Strasburg.  Translated  and 
edited  by  AUGUSTUS  A.  ESHNER,  M.  D.,  Professor  of  Clinical  Medicine, 
Philadelphia  Polyclinic.  Octavo  volume  of  440  pages,  fully  illustrated. 
Cloth,  $2.50  net. 

S.  Solis-Cohen,  M.  D., 

Professor  of  Clinical  Medicine,  Jefferson  Medical  College,  Philadelphia. 
"  I  consider  it  an  excellent  book.     I  have  recommended  it  in  speaking  to  my  students." 


Lehmann,  Neumann,  and 
Weaver's  Bacteriology 

Atlas  and  Epitome  of  Bacteriology :  INCLUDING  A  TEXT-BOOK  OF 
SPECIAL  BACTERIOLOGIC  DIAGNOSIS.  By  PROF.  DR.  K.  B.  LEHMANN 
and  DR.  R.  O.  NEUMANN,  of  Wiirzburg.  From  the  Second  Revised  and 
Enlarged  German  Edition.  Edited,  with  additions,  by  G.  H.  WEAVER, 
M.  D.,  Assistant  Professor  of  Pathology  and  Bacteriology,  Rush  Medical 
College,  Chicago.  In  two  parts.  Part  I. — 632  colored  figures  on  69 
lithographic  plates.  Part  II. — 51  i  pa^i-s  of  text,  illustrated.  Per  part: 
Cloth,  $2.50  net.  In  Saunders*  Hand-Atlas  Series. 


PATHOLOGY,    BACTERIOLOGY,    AND   PATHOLOGY.  15. 

Eyre's    Bacteriologic   Technique 

THE  ELEMENTS  OF  BACTERIOLOGIC  TECHNIQUE.     A  -Laboratory 

Guide  for  the  Medical,  Dental,  and  Technical  Student.     By  J.  W. 

H.  EYRE,  M.  D.,  F.  R.  S.  Edin.,  Lecturer  on  Bacteriology  at  the 

0        Medical  and  Dental  Schools,  London.     Octavo  of  375  pages,  with 

170  illustrations.     Cloth,  $2.50  net. 

American  Text-Book  of  Physiology  second  Edition 

AMERICAN  TEXT-BOOK  OF  PHYSIOLOGY.  In  two  volumes.  Edited  by 
WILLIAM  H.  HOWELL,  PH.  D.,  M.D.,  Professor  of  Physiology  in  the  Johns 
Hopkins  University,  Baltimore,  Md.  Two  royal  octavos  of  about  600 
pages  each,  illustrated.  Per  volume:  Cloth,  $3.00  net;  Half  Morocco, 
£4.25  net. 

"  The  work  will  stand  as  a  work  of  reference  on  physiology.  To  him  who  desires  to  know 
the  status  of  modern  physiology,  who  expects  to  obtain  suggestions  as  to  further  physio- 
logic inquiry,  we  know  of  none  in  English  which  so  eminently  meets  such  a  demand." — 
The  Medical  News. 

Warren's  Pathology  and  Therapeutics        second  Edition 

SURGICAL  PATHOLOGY  AND  THERAPEUTICS.  By  JOHN  COLLINS  WARREN, 
M.  D.,  LL.D.,  F.  R.  C.  S.  (Hon.),  Professor  of  Surgery,  Harvard  Med- 
ical School.  Octavo,  873  pages,  136  relief  and  lithographic  illustrations, 
33  in  colors.  With  an  Appendix  on  Scientific  Aids  to  Surgical  Diagnosis 
and  a  series  of  articles  on  Regional  Bacteriology.  Cloth,  $5.00  net; 
Half  Morocco,  $6.50  net. 

Gorham's  Bacteriology 

A  LABORATORY  COURSE  IN  BACTERIOLOGY.  For  the  Use  of  Medical, 
Agricultural,  and  Industrial  Students.  By  FREDERIC  P.  GORHAM,  A.  M.> 
Associate  Professor  of  Biology  in  Brown  University,  Providence,  R.  I.,, 
etc.  i2mo  of  192  pages,  with  97  illustrations.  Cloth,  $1.25  net. 

"  One  of  the  best  students'  laboratory  guides  to  the  study  of  bacteriology  on  the  mar- 
ket. .  .  .  The  technic  is  thoroughly  modern  and  amply  sufficient  for  all  practical  pur- 
poses."— American  Journal  of  the  Medical  Sciences. 

Raymond's  Physiology  New  (3<J)  Edition 

HUMAN  PHYSIOLOGY.  By  JOSEPH  H.  RAYMOND,  A.  M.,  M.  D.,  Pro- 
fessor of  Physiology  and  Hygiene,  Long  Island  College  Hospital,  New 
York.  Octavo  of  685  pages,  with  444  illustrations.  Cloth,  $3.50  net. 

"  The  book  is  well  gotten  up  and  well  printed,  and  may  be  regarded  as  a  trustworthy 
guide  for  the  student  and  a  useful  work  of  reference  for  the  general  practitioner.  The 
illustrations  are  numerous  and  are  well  executed." — The  Lancet,  London. 


16          BACTERIOLOGY,    PHYSIOLOGY,   AND  HISTOLOGY. 

Ball's    Bacteriology  Sixth  Edition,  Revised 

ESSENTIALS  OF  BACTERIOLOGY  :  being  a  concise  and  systematic  intro- 
duction to  the  Study  of  Micro-organisms.  By  M.  V.  BALL,  M.  D.,  Late 
Bacteriologist  to  St.  Agnes'  Hospital,  Philadelphia.  i2mo  of  289  pages, 
with  135  illustrations,  some  in  colors.  Cloth,  £1.00  net.  In  Saunters' 
Question-  Compend  Series. 

"  The  technic  with  regard  to  media,  staining,  mounting,  and  the  like  is  culled  from  the 
latest  authoritative  works."  —  The  Medical  Times,  New  York. 


Budgett's  Physiology  New  oa)  Edition 

ESSENTIALS  OF  PHYSIOLOGY.  Prepared  especially  for  Students  of  Medi- 
cine, and  arranged  with  questions  following  each  chapter.  By  SIDNEY 
P.  BUDGETT,  M.  D.,  formerly  Professor  of  Physiology,  Washington  Uni- 
versity, St.  Louis.  Revised  by  HAVAN  EMERSON,  M.  D.,  Demonstrator 
of  Physiology,  Columbia  University.  i2mo  volume  of  250  pages,  illus- 
trated. Cloth,  $  i  .  oo  net.  Saundcrs*  Question-  Compend  Series. 

"He  has  an  excellent  conception  of  his  subject.  .  .  It  is  one  of  the  most  satisfactory 
books  of  this  class"  —  University  of  Pennsylvania  Medical  Bulletin. 

Leroy's  Histology  New  (4th)  Edition 

ESSENTIALS  OF  HISTOLOGY.  By  Louis  LEROY,  M.  D.,  Professor  of 
Histology  and  Pathology,  Vanderbilt  University,  Nashville,  Tennessee. 
i2mo,  263  pages,  with  92  original  illustrations.  Cloth,  $1.00  net.  In 
Saunters'  Question-  Compend  Series. 

"  The  work  in  its  present  form  stands  as  a  model  of  what  a  student's  aid  should  be  ;  and 
we  unhesitatingly  say  that  the  practitioner  as  well  would  find  a  glance  through  the  book 
of  lasting  benefit."  —  The  Medical  World,  Philadelphia. 

Barton  and  Wells'  Medical  Thesaurus 

A  THESAURUS  OF  MEDICAL  WORDS  AND  PHRASES.  By  WILFRED  M. 
BARTON,  M.  D.,  Assistant  Professor  of  Materia  Medica  and  Therapeutics, 
and  WALTER  A.  WELLS,  M.D.,  Demonstrator  of  Laryngology,  Georgetown 
University,  Washington,  D.  C.  i2mo,  534  pages.  Flexible  leather, 
$2.50  net;  thumb  indexed,  $3.00  net. 

American  Pocket  Dictionary  New  (TthTcrtTion 

BORLAND'S  POCKET  MEDICAL  DICTIONARY.  Edited  by  W.  A.  NEW- 
MAN DORLAND,  M.  D.,  Editor  "  American  Illustrated  Medical  Dic- 
tionary." Containing  the  pronunciation  and  definition  of  the  principal 
words  used  in  medicine  and  kindred  sciences,  with  64  extensive  tables. 
6  10  pages.  Flexible  leather,  with  gold  edges,  £1.00  net;  with  patent 
thumb  index,  $1.25  net. 

"  I  can  recommend  it  to  our  students  without  reserve."  —  J.  H.  HOLLAND,  M.  D.,  of 
the  Je/erson  Medical  College,  Philadelphia. 


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